Methods of treating prlr positive breast cancer

ABSTRACT

Provided herein are methods of treating prolactin receptor positive breast cancer using an antibody drug conjugate (ADC) comprising an anti-PRLR antibody or antigen-binding fragment thereof conjugated to a cytotoxic agent. In certain embodiments, the anti-PRLR antibody or antigen-binding fragment thereof is conjugated to maytansinoid. In certain embodiments, the method of treating PRLR positive breast cancer includes administering the ADC in combination with one or more chemotherapeutic agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 34 U.S.C. § 119(e) of U.S.Provisional Application No. 62/427,534, filed Nov. 29, 2016, which isherein specifically incorporated by reference in its entirety.

FIELD

The present disclosure is related to methods of treating PRLR positivebreast cancer using (a) an antibody-drug conjugate comprising ananti-PRLR antibody conjugated to a cytotoxic agent and (b) fulvestrant.

SEQUENCE LISTING

An official copy of the sequence listing is submitted concurrently withthe specification electronically via EFS-Web as an ASCII formattedsequence listing with a file name of “10314P1-US_SeqListing.txt”, acreation date of Nov. 29, 2016, and a size of about 144 KB. The sequencelisting contained in this ASCII formatted document is part of thespecification and is herein incorporated by reference in its entirety.

BACKGROUND

Prolactin is a polypeptide growth hormone that exerts its activity byinteracting with the prolactin receptor (PRLR). PRLR is a singletransmembrane receptor belonging to the class 1 cytokine receptorsuperfamily. The binding of prolactin to PRLR leads to receptordimerization and intracellular signaling. Signaling through PRLR isassociated with various processes such as mammary gland development,lactation, reproduction and immunomodulation. Moreover, high levels ofPRLR expression have been detected in breast, prostate, and other tumortypes.

Blockade of PRLR signaling has been suggested as a means for treatingbreast and prostate cancer. (See, e.g., Damiano and Wasserman, April2013, Clin. Cancer Res. 19(7):1644-1650). Anti-PRLR antibodies arementioned, e.g., in U.S. Pat. Nos. 9,302,015, 7,867,493, and 7,422,899.Nonetheless, there is a need in the art for novel methods of treatingcancer and other disorders associated with PRLR expression and/orsignaling.

The present disclosure is directed toward overcoming one or more of theproblems discussed above.

BRIEF SUMMARY

Provided herein are antibody-drug conjugates (ADCs) comprising fullyhuman monoclonal anti-PRLR antibodies, and antigen-binding fragmentsthereof, conjugated to a cytotoxic agent. The ADCs can be useful totreat PRLR positive breast cancer, and are particularly useful when usedas a combination therapy with fulvestrant.

In one aspect, provided is a method of treating PRLR positive breastcancer, comprising co-administering to a patient a therapeuticallyeffective amount of: (a) an antibody-drug conjugate (ADC) comprising anantibody or antigen-binding fragment thereof conjugated to a cytotoxicagent, wherein the antibody or antigen-binding fragment thereof binds tohuman prolactin receptor (PRLR), and (b) fulvestrant.

In some embodiments, the cytotoxic agent is a maytansinoid.

In another aspect, provided is a method of treating PRLR positive breastcancer in a patient, comprising co-administering to the patient atherapeutically effective amount of: (a) an antibody-drug conjugate(ADC) comprising an antibody or antigen-binding fragment thereofconjugated to maytansinoid, wherein the antibody or antigen-bindingfragment thereof binds to human prolactin receptor (PRLR), and (b)fulvestrant.

In some embodiments, the maytansinoid is DM1 or DM4.

In some embodiments, the ADC kills cells that express PRLR at anexpression level of less than 30-fold above background, for example, atan expression level of less than 20-fold above background. In someembodiments, the ADC kills cells that express PRLR at an expressionlevel greater than 12-fold above background but less than 30-fold abovebackground.

In another aspect, provided is a method of treating PRLR positive breastcancer in a patient, comprising co-administering to the patient atherapeutically effective amount of: (a) an antibody-drug conjugate(ADC) comprising an antibody or antigen-binding fragment thereofconjugated to DM1 through an MCC linker, wherein the antibody orantigen-binding fragment thereof binds to human prolactin receptor(PRLR), and (b) fulvestrant.

In some embodiments, the patient was previously treated with an estrogenreceptor inhibitor.

Treatment of a PRLR positive breast cancer with an ADC described hereincan have one or more of the following effects, among others: the ADC caninhibit prolactin mediated STAT5 activity; the ADC can induce mitoticarrest in cells expressing PRLR; and/or the ADC can inhibit the growthof cells expressing PRLR.

In another aspect, provided herein is a method for killing tumor cellsthat express low levels of PRLR, the method comprising contacting thecells with: (a) an antibody-drug conjugate (ADC) comprising an antibodyor antigen-binding fragment thereof conjugated to a cytotoxic agent,wherein the antibody or antigen-binding fragment thereof binds to humanprolactin receptor (PRLR), and (b) fulvestrant. In some embodiments, thetumor cells express less than 1 million copies of PRLR per cell. In someembodiments, the tumor cells express between 300 and 500,000 copies ofPRLR per cell.

In another aspect, provided herein is a method for killing tumor cellsthat express low levels of PRLR, the method comprising contacting thecells with: (a) an antibody-drug conjugate (ADC) comprising an antibodyor antigen-binding fragment thereof conjugated to a maytansinoid,wherein the antibody or antigen-binding fragment thereof binds to humanprolactin receptor (PRLR), and (b) fulvestrant.

The antibodies useful according to the methods provided herein can befull-length, for example, an IgG1 or and IgG4 antibody, or may compriseonly an antigen-binding portion, for example, a Fab, F(ab′)₂, or scFvfragment, and can be modified to affect functionality, e.g., toeliminate residual effector functions (Reddy et al., (2000), J. Immunol.164: 1925-1933).

Provided herein are methods of using ADC comprising antibodies andantigen-binding fragments thereof that bind human prolactin receptor(PRLR). The ADCs are useful, inter alia, for targeting tumor cells thatexpress PRLR.

Exemplary anti-PRLR antibodies useful herein are listed in Tables 1 and2. Table 1 sets forth the amino acid sequence identifiers of the heavychain variable regions (HCVRs), light chain variable regions (LCVRs),heavy chain complementarity determining regions (HCDR1, HCDR2 andHCDR3), and light chain complementarity determining regions (LCDR1,LCDR2 and LCDR3) of the exemplary anti-PRLR antibodies. Table 2 setsforth the nucleic acid sequence identifiers of the HCVRs, LCVRs, HCDR1,HCDR2 HCDR3, LCDR1, LCDR2 and LCDR3 of the exemplary anti-PRLRantibodies.

The ADCs useful herein comprise antibodies or antigen-binding fragmentsthereof that specifically bind PRLR, comprising an HCVR comprising anamino acid sequence selected from any of the HCVR amino acid sequenceslisted in Table 1, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The ADCs useful herein comprise antibodies or antigen-binding fragmentsthereof that specifically bind PRLR, comprising an LCVR comprising anamino acid sequence selected from any of the LCVR amino acid sequenceslisted in Table 1, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The ADCs useful herein comprise antibodies or antigen-binding fragmentsthereof that specifically bind PRLR, comprising an HCVR and an LCVRamino acid sequence pair (HCVR/LCVR) comprising any of the HCVR aminoacid sequences listed in Table 1 paired with any of the LCVR amino acidsequences listed in Table 1. According to certain embodiments, the ADCsuseful herein comprise antibodies, or antigen-binding fragments thereof,comprising an HCVR/LCVR amino acid sequence pair contained within any ofthe exemplary anti-PRLR antibodies listed in Table 1. In certainembodiments, the HCVR/LCVR amino acid sequence pair is selected from thegroup consisting of: 18/26; 66/74; 274/282; 290/298; and 370/378.

The ADCs useful herein comprise antibodies or antigen-binding fragmentsthereof that specifically bind PRLR, comprising a heavy chain CDR1(HCDR1) comprising an amino acid sequence selected from any of the HCDR1amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The ADCs useful herein comprise antibodies or antigen-binding fragmentsthereof that specifically bind PRLR, comprising a heavy chain CDR2(HCDR2) comprising an amino acid sequence selected from any of the HCDR2amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The ADCs useful herein comprise antibodies or antigen-binding fragmentsthereof that specifically bind PRLR, comprising a heavy chain CDR3(HCDR3) comprising an amino acid sequence selected from any of the HCDR3amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The ADCs useful herein comprise antibodies or antigen-binding fragmentsthereof that specifically bind PRLR, comprising a light chain CDR1(LCDR1) comprising an amino acid sequence selected from any of the LCDR1amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The ADCs useful herein comprise antibodies or antigen-binding fragmentsthereof that specifically bind PRLR, comprising a light chain CDR2(LCDR2) comprising an amino acid sequence selected from any of the LCDR2amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The ADCs useful herein comprise antibodies or antigen-binding fragmentsthereof that specifically bind PRLR, comprising a light chain CDR3(LCDR3) comprising an amino acid sequence selected from any of the LCDR3amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The ADCs useful herein comprise antibodies or antigen-binding fragmentsthereof that specifically bind PRLR, comprising an HCDR3 and an LCDR3amino acid sequence pair (HCDR3/LCDR3) comprising any of the HCDR3 aminoacid sequences listed in Table 1 paired with any of the LCDR3 amino acidsequences listed in Table 1. According to certain embodiments, the ADCsuseful herein comprise antibodies, or antigen-binding fragments thereof,comprising an HCDR3/LCDR3 amino acid sequence pair contained within anyof the exemplary anti-PRLR antibodies listed in Table 1. In certainembodiments, the HCDR3/LCDR3 amino acid sequence pair is selected fromthe group consisting of: 24/32; 72/80; 280/288; 296/304; and 376/384.

The ADCs useful herein comprise antibodies or antigen-binding fragmentsthereof that specifically bind PRLR, comprising a set of six CDRs (i.e.,HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of theexemplary anti-PRLR antibodies listed in Table 1. In certainembodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acidsequences set is selected from the group consisting of:20-22-24-28-30-32; 68-70-72-76-78-80; 276-278-280-284-286-288;292-294-296-300-302-304; and 372-374-376-380-382-384.

In a related embodiment, the ADCs useful herein comprise antibodies, orantigen-binding fragments thereof that specifically bind PRLR,comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3)contained within an HCVR/LCVR amino acid sequence pair as defined by anyof the exemplary anti-PRLR antibodies listed in Table 1. For example,the antibodies or antigen-binding fragments thereof that specificallybind PRLR, comprise the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acidsequences set contained within an HCVR/LCVR amino acid sequence pairselected from the group consisting of: 18/26; 66/74; 274/282; 290/298;and 370/378. Methods and techniques for identifying CDRs within HCVR andLCVR amino acid sequences are well known in the art and can be used toidentify CDRs within the specified HCVR and/or LCVR amino acid sequencesdisclosed herein. Exemplary conventions that can be used to identify theboundaries of CDRs include, e.g., the Kabat definition, the Chothiadefinition, and the AbM definition. In general terms, the Kabatdefinition is based on sequence variability, the Chothia definition isbased on the location of the structural loop regions, and the AbMdefinition is a compromise between the Kabat and Chothia approaches.See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,”National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al.,J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad.Sci. USA 86:9268-9272 (1989). Public databases are also available foridentifying CDR sequences within an antibody.

Also provided are nucleic acid molecules encoding anti-PRLR antibodiesor portions thereof, which are useful in the methods described herein.For example, nucleic acid molecules are provided encoding any of theHCVR amino acid sequences listed in Table 1; in certain embodiments thenucleic acid molecule comprises a polynucleotide sequence selected fromany of the HCVR nucleic acid sequences listed in Table 2, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity thereto. In anotherexample, nucleic acid molecules are provided encoding any of the LCVRamino acid sequences listed in Table 1; in certain embodiments thenucleic acid molecule comprises a polynucleotide sequence selected fromany of the LCVR nucleic acid sequences listed in Table 2, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity thereto.

Also provided herein are nucleic acid molecules encoding any of theHCDR1 amino acid sequences listed in Table 1; in certain embodiments thenucleic acid molecule comprises a polynucleotide sequence selected fromany of the HCDR1 nucleic acid sequences listed in Table 2, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity thereto.

Also provided herein are nucleic acid molecules encoding any of theHCDR2 amino acid sequences listed in Table 1; in certain embodiments thenucleic acid molecule comprises a polynucleotide sequence selected fromany of the HCDR2 nucleic acid sequences listed in Table 2, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity thereto.

Also provided herein are nucleic acid molecules encoding any of theHCDR3 amino acid sequences listed in Table 1; in certain embodiments thenucleic acid molecule comprises a polynucleotide sequence selected fromany of the HCDR3 nucleic acid sequences listed in Table 2, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity thereto.

Also provided herein are nucleic acid molecules encoding any of theLCDR1 amino acid sequences listed in Table 1; in certain embodiments thenucleic acid molecule comprises a polynucleotide sequence selected fromany of the LCDR1 nucleic acid sequences listed in Table 2, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity thereto.

Also provided herein are nucleic acid molecules encoding any of theLCDR2 amino acid sequences listed in Table 1; in certain embodiments thenucleic acid molecule comprises a polynucleotide sequence selected fromany of the LCDR2 nucleic acid sequences listed in Table 2, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity thereto.

Also provided herein are nucleic acid molecules encoding any of theLCDR3 amino acid sequences listed in Table 1; in certain embodiments thenucleic acid molecule comprises a polynucleotide sequence selected fromany of the LCDR3 nucleic acid sequences listed in Table 2, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity thereto.

Also provided herein are nucleic acid molecules encoding an HCVR,wherein the HCVR comprises a set of three CDRs (i.e.,HCDR1-HCDR2-HCDR3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequenceset is as defined by any of the exemplary anti-PRLR antibodies listed inTable 1.

Also provided herein are nucleic acid molecules encoding an LCVR,wherein the LCVR comprises a set of three CDRs (i.e.,LCDR1-LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequenceset is as defined by any of the exemplary anti-PRLR antibodies listed inTable 1.

Also provided herein are nucleic acid molecules encoding both an HCVRand an LCVR, wherein the HCVR comprises an amino acid sequence of any ofthe HCVR amino acid sequences listed in Table 1, and wherein the LCVRcomprises an amino acid sequence of any of the LCVR amino acid sequenceslisted in Table 1. In certain embodiments, the nucleic acid moleculecomprises a polynucleotide sequence selected from any of the HCVRnucleic acid sequences listed in Table 2, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity thereto, and a polynucleotide sequenceselected from any of the LCVR nucleic acid sequences listed in Table 2,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity thereto. Incertain embodiments, the nucleic acid molecule encodes an HCVR and LCVR,wherein the HCVR and LCVR are both derived from the same anti-PRLRantibody listed in Table 1.

Also provided herein are recombinant expression vectors capable ofexpressing a polypeptide comprising a heavy or light chain variableregion of an anti-PRLR antibody. For example, the recombinant expressionvectors can comprise any of the nucleic acid molecules mentioned above,i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and/or CDRsequences as set forth in Table 1. Also provided herein are host cellsinto which such vectors have been introduced, as well as methods ofproducing the antibodies or portions thereof by culturing the host cellsunder conditions permitting production of the antibodies or antibodyfragments, and recovering the antibodies and antibody fragments soproduced.

In some embodiments, the ADCs useful herein comprise anti-PRLRantibodies having a modified glycosylation pattern. In some aspects,modification to remove undesirable glycosylation sites may be useful, oran antibody lacking a fucose moiety present on the oligosaccharidechain, for example, to increase antibody dependent cellular cytotoxicity(ADCC) function (see Shield et al. (2002) JBC 277:26733). In otherapplications, modification of galactosylation can be made in order tomodify complement dependent cytotoxicity (CDC).

In another aspect, the disclosure provides a pharmaceutical compositioncomprising an ADC, wherein the ADC comprises a recombinant humanantibody or fragment thereof which specifically binds PRLR conjugated tocytotoxic agent, and a pharmaceutically acceptable carrier. In a relatedaspect, the composition is an ADC comprising combination of an anti-PRLRantibody conjugated to a cytotoxic agent, and a second therapeuticagent. In one embodiment, the second therapeutic agent is any agent thatis advantageously combined with an anti-PRLR antibody, for example,fulvestrant. The ADCs can comprise an anti-PRLR antibody conjugated to acytotoxic agent, for example, a maytansinoid. In some embodiments, themaytansinoid is DM1 or DM4. Exemplary combination therapies,co-formulations, and ADCs involving the anti-PRLR antibodies useful inthe methods provided herein are disclosed elsewhere in the presentdisclosure.

In yet another aspect, provided are therapeutic methods for killingtumor cells or for inhibiting or attenuating tumor cell growth using ananti-PRLR antibody or antigen-binding portion of an antibody as providedherein. The therapeutic methods comprise administering a therapeuticallyeffective amount of a pharmaceutical composition comprising an ADCdescribed herein to a subject having PRLR-positive breast cancer, inaddition to treatment with fulvestrant.

The ADCs useful herein comprise anti-PRLR antibodies that interact withone or more amino acids contained within the sequenceMHECPDYITGGPNSCHFGKQYTSMWRTYIMM (SEQ ID NO:405, corresponding to aminoacids 72 to 102 of SEQ ID NO:404).

Provided herein is a peptide that comprises an amino acid sequence thatis the same as that of a fragment having no more than 102 amino acids ofthe amino acid sequence of SEQ. ID NO.:404. In a related aspect, thefragment is within the first fibronectin-like type III domain of theextracellular domain of PRLR (amino acids 27-128 of SEQ ID NO:404). Alsoprovided is a peptide consisting essentially of an amino acid sequencethat is the same as that of a fragment having no more than 102 aminoacids of the amino acid sequence of SEQ. ID NO.:404. In a relatedaspect, the fragment is within the first fibronectin-like type IIIdomain of the extracellular domain of PRLR (amino acids 27-128 of SEQ IDNO:404). Further provided is a peptide that consists of an amino acidsequence that is the same as that of a fragment having no more than 102amino acids of the amino acid sequence of SEQ. ID NO.:404. In a relatedaspect, the fragment is within the first fibronectin-like type IIIdomain of the extracellular domain of PRLR (amino acids 27-128 of SEQ IDNO:404). In another related aspect, the fragment is selected from thegroup consisting of (a) amino acids 72 to 94 of SEQ ID NO:404; (b) aminoacids 72 to 95 of SEQ ID NO:404; (c) amino acids 96 to 101 of SEQ IDNO:404; and (d) amino acids 96 to 102 of SEQ ID NO:404. The peptide canbe manufactured synthetically or isolated.

Also provided herein is a composition comprising a peptide thatcomprises an amino acid sequence that is the same as that of a fragmenthaving no more than 102 amino acids of the amino acid sequence of SEQ.ID NO.:404. In a related aspect, the fragment is within the firstfibronectin-like type III domain of the extracellular domain of PRLR(amino acids 27-128 of SEQ ID NO:404). Also provided herein are methodsof detecting the presence and/or quantity of specific antibodies to thepeptide in a sample, the method comprising contacting the sample withthe composition, and detecting the presence and/or measuring thequantity of the specific antibodies bound to the composition. In arelated aspect, the peptide is bound to a solid support. In anotherrelated aspect, the peptide is labeled such as with an enzyme,florescent, biotin, or radioactive label.

Also provided herein is a nucleic acid sequence encoding an amino acidsequence that is the same as that of a fragment having no more than 102amino acids of the amino acid sequence of SEQ. ID NO.:404. In a relatedaspect, the fragment is within the first fibronectin-like type IIIdomain of the extracellular domain of PRLR (amino acids 27-128 of SEQ IDNO:404).

Also provided herein is a peptide that comprises an amino acid sequenceof SEQ. ID NO.:405. Also provided is a peptide that consists essentiallyof an amino acid sequence of SEQ. ID NO.:405. Further provided is apeptide that consists of an amino acid sequence SEQ. ID NO.:405. Thepeptide can be manufactured synthetically or isolated.

Other embodiments will become apparent from a review of the ensuingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B demonstrates H1H6958N2 and H1H6958N2-DM1 blocking PRLRbinding to prolactin (PRL). Human (A) or monkey (B) PRLR bound tocaptured PRL with EC₅₀ values of 4.9 nM and 5.7 nM, respectively (solidsquares in insets in A and B). H1H6958N2-DM1 (solid circles) andH1H6958N2 (open circles) blocked human and monkey PRLR binding to PRL.Specifically, H1H6958N2-DM1 and H1H6958N2 blocked binding of 10 nM humanPRLR to PRL with IC₅₀ values of 4.4 nM and 5.0 nM, respectively (A).Similarly, H1H6958N2-DM1 and H1H6958N2 blocked binding of 10 nM monkeyPRLR to PRL with IC₅₀ values of 4.2 nM and 5.8 nM, respectively (B).Both control ADC (solid triangles) and control mAb (open triangles)displayed no blockade under identical assay conditions (A, B). Molarityindicates antibody concentration for ADCs and mAbs. Error bars representSD.

FIG. 1C shows HEK293/PRLR/STAT5-Luc cells treated with increasingconcentrations of PRL (stars) resulting in an increase of luminescence(plotted as RLU), indicative of PRLR signaling-mediated induction ofluciferase expression (C). In the presence of a constant concentrationof 2 nM PRL, increasing concentrations (3.3 pM to 200 nM) ofH1H6958N2-DM1 (closed circles) or H1H6958N2 (open circles) blockedPRLR-driven luciferase expression with IC₅₀ values of 0.4 nM for bothantibodies. The non-binding control ADC (closed triangles) and theunconjugated control mAb (gray, open triangles) did not blockPRL-mediated PRLR signaling under identical assay conditions. Mean andSD data is shown. RLU: relative light units. The x-axis indicates themolar concentration of ADC or mAb except for the PRL dose responsecurve, where molar concentration of PRL is indicated.

FIGS. 2A, 2B, 2C, and 2D demonstrate H1H6958N2-DM1 is active againstMCF7 tumors and MCF7/PRLR tumors in vivo. NCr Nude mice bearingestablished MCF7 or MCF7/PRLR tumors were treated with H1H6958N2-DM1 andcontrols. The line graphs depict the average tumor volume ±SEM per groupover time. The arrow indicates the day of dosing. Results of a MCF7single dose study (A) and results of a MCF7 multi-dose (q7dx3) study(B). Results of a MCF7/PRLR single dose study (C) and results of aMCF7/PRLR multi-dose (q7dx3) study (D).

FIGS. 3A, 3B, and 3C demonstrate H1H6958N2-DM1 is efficacious against inT47Dv11 tumors and has combined activity with fulvestrant. SCID micebearing established T47Dv11 tumors were treated with H1H6958N2-DM1 andcontrols. T47Dv11 is a tumorigenic variant of parental T47D cellsgenerated through in vivo passaging. The line graphs depict the averagetumor volume ±SEM per group over time. Results of a single dose study(A) and results of a multi-dose (q7dx3) study (B). Results of the singledose H1H6958N2-DM1 and fulvestrant combination study (C).

FIG. 4 provides activity of weekly fulvestrant treatment of T47DvIIxenografts.

FIG. 5 demonstrates H1H6958N2-DM1 mediates mitotic arrest in T47Dv11tumors. IHC for phospho-Histone H3 (PHH3) was used as a marker of cellsaccumulating in a mitotic state. The percent of PHH3 positive nucleiwere quantified for each treatment.

FIGS. 6A and 6B demonstrate H1H6958N2-DM1 is active in TM00107 PDX(Jackson Labs) tumors. NSG mice bearing established TM00107 tumors weretreated with PRLR ADC and controls. The line graphs depict the averagetumor volume ±SEM per group over time. The arrow indicates the day ofdosing. Results of a multi-dose (q7dx4) study (A) and the change intumor volume at Day 30 in the multi-dose study (B).

DESCRIPTION

Before the present compositions and methods are described, it is to beunderstood that this disclosure is not limited to particularcompositions and methods, and experimental conditions described, as suchmethods, compositions, and conditions may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentlydisclosed subject matter, exemplary methods and materials are nowdescribed.

Definitions

The expression prolactin receptor, “PRLR,” and the like, as used herein,refers to the human prolactin receptor, comprising the amino acidsequence as set forth in SEQ ID NO:404. The expression “PRLR” includesboth monomeric and multimeric PRLR molecules. As used herein, theexpression “monomeric human PRLR” means a PRLR protein or portionthereof that does not contain or possess any multimerizing domains andthat exists under normal conditions as a single PRLR molecule without adirect physical connection to another PRLR molecule. An exemplarymonomeric PRLR molecule is the molecule referred to herein as“hPRLR.mmh” comprising the amino acid sequence of SEQ ID NO:401 (see,e.g., Example 3, herein). As used herein, the expression “dimeric humanPRLR” means a construct comprising two PRLR molecules connected to oneanother through a linker, covalent bond, non-covalent bond, or through amultimerizing domain such as an antibody Fc domain. An exemplary dimericPRLR molecule is the molecule referred to herein as “hPRLR.mFc”comprising the amino acid sequence of SEQ ID NO:402 (see, e.g., Example3, herein).

All references to proteins, polypeptides and protein fragments hereinare intended to refer to the human version of the respective protein,polypeptide or protein fragment unless explicitly specified as beingfrom a non-human species. Thus, the expression “PRLR” means human PRLRunless specified as being from a non-human species, e.g., “mouse PRLR,”“monkey PRLR,” etc.

As used herein, the expression “cell surface-expressed PRLR” means oneor more PRLR protein(s), or the extracellular domain thereof, thatis/are expressed on the surface of a cell in vitro or in vivo, such thatat least a portion of a PRLR protein is exposed to the extracellularside of the cell membrane and is accessible to an antigen-bindingportion of an antibody. A “cell surface-expressed PRLR” can comprise orconsist of a PRLR protein expressed on the surface of a cell whichnormally expresses PRLR protein. Alternatively, “cell surface-expressedPRLR” can comprise or consist of PRLR protein expressed on the surfaceof a cell that normally does not express human PRLR on its surface buthas been artificially engineered to express PRLR on its surface.

As used herein, the expression “anti-PRLR antibody” includes bothmonovalent antibodies with a single specificity, as well as bispecificantibodies comprising a first arm that binds PRLR and a second arm thatbinds a second (target) antigen, wherein the anti-PRLR arm comprises anyof the HCVR/LCVR or CDR sequences as set forth in Table 1 herein. Theexpression “anti-PRLR antibody” also includes antibody-drug conjugates(ADCs) comprising an anti-PRLR antibody or antigen-binding portionthereof conjugated to a drug or toxin (i.e., cytotoxic agent). Theexpression “anti-PRLR antibody” also includes antibody-radionuclideconjugates (ARCs) comprising an anti-PRLR antibody or antigen-bindingportion thereof conjugated to a radionuclide.

The term “antibody”, as used herein, means any antigen-binding moleculeor molecular complex comprising at least one complementarity determiningregion (CDR) that specifically binds to or interacts with a particularantigen (e.g., PRLR). The term “antibody” includes immunoglobulinmolecules comprising four polypeptide chains, two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds, as well asmultimers thereof (e.g., IgM). Each heavy chain comprises a heavy chainvariable region (abbreviated herein as HCVR or V_(H)) and a heavy chainconstant region. The heavy chain constant region comprises threedomains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises a lightchain variable region (abbreviated herein as LCVR or V_(L)) and a lightchain constant region. The light chain constant region comprises onedomain (C_(L)1). The V_(H) and V_(L) regions can be further subdividedinto regions of hypervariability, termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FR). Each V_(H) and V_(L) is composed of threeCDRs and four FRs, arranged from amino-terminus to carboxy-terminus inthe following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In differentembodiments, the FRs of the anti-PRLR antibody (or antigen-bindingportion thereof) may be identical to the human germline sequences, ormay be naturally or artificially modified. An amino acid consensussequence may be defined based on a side-by-side analysis of two or moreCDRs.

The term “antibody”, as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments;

-   -   (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fv        fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb        fragments; and (vii) minimal recognition units consisting of the        amino acid residues that mimic the hypervariable region of an        antibody (e.g., an isolated complementarity determining region        (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4        peptide. Other engineered molecules, such as domain-specific        antibodies, single domain antibodies, domain-deleted antibodies,        chimeric antibodies, CDR-grafted antibodies, diabodies,        triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent        nanobodies, bivalent nanobodies, etc.), small modular        immunopharmaceuticals (SMIPs), and shark variable IgNAR domains,        are also encompassed within the expression “antigen-binding        fragment,” as used herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody include: (i) V_(H)-C_(H)1; (ii) V_(H)-C_(H)2; (iii)V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v) V_(H)-C_(H)1-C_(H)2-C_(H)3;(vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L); (viii) V_(L)-C_(H)1; (ix)V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi) V_(L)-C_(H)1-C_(H)2; (xii)V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii) V_(L)-C_(H)2-C_(H)3; and (xiv)V_(L)-C_(L). In any configuration of variable and constant domains,including any of the exemplary configurations listed above, the variableand constant domains may be either directly linked to one another or maybe linked by a full or partial hinge or linker region. A hinge regionmay consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) aminoacids which result in a flexible or semi-flexible linkage betweenadjacent variable and/or constant domains in a single polypeptidemolecule. Moreover, an antigen-binding fragment of an antibody providedherein may comprise a homo-dimer or hetero-dimer (or other multimer) ofany of the variable and constant domain configurations listed above innon-covalent association with one another and/or with one or moremonomeric V_(H) or V_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present disclosure using routine techniques available in the art.

The antibodies useful herein may function through complement-dependentcytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity(ADCC). “Complement-dependent cytotoxicity” (CDC) refers to lysis ofantigen-expressing cells by an antibody in the presence of complement.“Antibody-dependent cell-mediated cytotoxicity” (ADCC) refers to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and thereby leadto lysis of the target cell. CDC and ADCC can be measured using assaysthat are well known and available in the art. (See, e.g., U.S. Pat. Nos.5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci.(USA) 95:652-656). The constant region of an antibody is important inthe ability of an antibody to fix complement and mediate cell-dependentcytotoxicity. Thus, the isotype of an antibody may be selected on thebasis of whether it is desirable for the antibody to mediatecytotoxicity.

In certain embodiments, the anti-PRLR antibodies provided herein arehuman antibodies. The term “human antibody”, as used herein, is intendedto include antibodies having variable and constant regions derived fromhuman germline immunoglobulin sequences. The human antibodies mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs and in particular CDR3. However, the term “human antibody”, as usedherein, is not intended to include antibodies in which CDR sequencesderived from the germline of another mammalian species, such as a mouse,have been grafted onto human framework sequences.

The antibodies may, in some embodiments, be recombinant humanantibodies. The term “recombinant human antibody”, as used herein, isintended to include all human antibodies that are prepared, expressed,created or isolated by recombinant means, such as antibodies expressedusing a recombinant expression vector transfected into a host cell(described further below), antibodies isolated from a recombinant,combinatorial human antibody library (described further below),antibodies isolated from an animal (e.g., a mouse) that is transgenicfor human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl.Acids Res. 20:6287-6295) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the V_(H) andV_(L) regions of the recombinant antibodies are sequences that, whilederived from and related to human germline V_(H) and V_(L) sequences,may not naturally exist within the human antibody germline repertoire invivo.

Human antibodies can exist in two forms that are associated with hingeheterogeneity. In one form, an immunoglobulin molecule comprises astable four chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via inter-chain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a covalentlycoupled light and heavy chain (half-antibody). These forms have beenextremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et al.(1993) Molecular Immunology 30:105) to levels typically observed using ahuman IgG1 hinge. The instant disclosure provides antibodies having oneor more mutations in the hinge, C_(H)2 or C_(H)3 region which may bedesirable, for example, in production, to improve the yield of thedesired antibody form.

The antibodies provided herein may be isolated antibodies. An “isolatedantibody,” as used herein, means an antibody that has been identifiedand separated and/or recovered from at least one component of itsnatural environment. For example, an antibody that has been separated orremoved from at least one component of an organism, or from a tissue orcell in which the antibody naturally exists or is naturally produced, isan “isolated antibody” for purposes of the present disclosure. Anisolated antibody also includes an antibody in situ within a recombinantcell. Isolated antibodies are antibodies that have been subjected to atleast one purification or isolation step. According to certainembodiments, an isolated antibody may be substantially free of othercellular material and/or chemicals.

The anti-PRLR antibodies useful in the methods disclosed herein maycomprise one or more amino acid substitutions, insertions and/ordeletions in the framework and/or CDR regions of the heavy and lightchain variable domains as compared to the corresponding germlinesequences from which the antibodies were derived. Such mutations can bereadily ascertained by comparing the amino acid sequences disclosedherein to germline sequences available from, for example, publicantibody sequence databases. The present disclosure includes antibodies,and antigen-binding fragments thereof, which are derived from any of theamino acid sequences disclosed herein, wherein one or more amino acidswithin one or more framework and/or CDR regions are mutated to thecorresponding residue(s) of the germline sequence from which theantibody was derived, or to the corresponding residue(s) of anotherhuman germline sequence, or to a conservative amino acid substitution ofthe corresponding germline residue(s) (such sequence changes arereferred to herein collectively as “germline mutations”). A person ofordinary skill in the art, starting with the heavy and light chainvariable region sequences disclosed herein, can easily produce numerousantibodies and antigen-binding fragments which comprise one or moreindividual germline mutations or combinations thereof. In certainembodiments, all of the framework and/or CDR residues within the V_(H)and/or V_(L) domains are mutated back to the residues found in theoriginal germline sequence from which the antibody was derived. In otherembodiments, only certain residues are mutated back to the originalgermline sequence, e.g., only the mutated residues found within thefirst 8 amino acids of FR1 or within the last 8 amino acids of FR4, oronly the mutated residues found within CDR1, CDR2 or CDR3. In otherembodiments, one or more of the framework and/or CDR residue(s) aremutated to the corresponding residue(s) of a different germline sequence(i.e., a germline sequence that is different from the germline sequencefrom which the antibody was originally derived). Furthermore, theantibodies of the present disclosure may contain any combination of twoor more germline mutations within the framework and/or CDR regions,e.g., wherein certain individual residues are mutated to thecorresponding residue of a particular germline sequence while certainother residues that differ from the original germline sequence aremaintained or are mutated to the corresponding residue of a differentgermline sequence. Once obtained, antibodies and antigen-bindingfragments that contain one or more germline mutations can be easilytested for one or more desired property such as, improved bindingspecificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. Antibodies and antigen-binding fragmentsobtained in this general manner are encompassed within the presentdisclosure.

Also provided herein are methods of treatment using ADCs comprisinganti-PRLR antibodies, where the antibodies comprise variants of any ofthe HCVR, LCVR, and/or CDR amino acid sequences disclosed herein havingone or more conservative substitutions. For example, the anti-PRLRantibodies can have HCVR, LCVR, and/or CDR amino acid sequences with,e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservativeamino acid substitutions relative to any of the HCVR, LCVR, and/or CDRamino acid sequences set forth in Table 1 herein.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95%, and more preferablyat least about 96%, 97%, 98% or 99% of the nucleotide bases, as measuredby any well-known algorithm of sequence identity, such as FASTA, BLASTor Gap, as discussed below. A nucleic acid molecule having substantialidentity to a reference nucleic acid molecule may, in certain instances,encode a polypeptide having the same or substantially similar amino acidsequence as the polypeptide encoded by the reference nucleic acidmolecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 95% sequence identity, even more preferably atleast 98% or 99% sequence identity. Preferably, residue positions whichare not identical differ by conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See, e.g., Pearson (1994)Methods Mol. Biol. 24: 307-331, herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include (1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains:serine and threonine; (3) amide-containing side chains: asparagine andglutamine; (4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; (5) basic side chains: lysine, arginine, and histidine; (6)acidic side chains: aspartate and glutamate, and (7) sulfur-containingside chains are cysteine and methionine. Preferred conservative aminoacids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443-1445, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence to a database containing a large number of sequences fromdifferent organisms is the computer program BLAST, especially BLASTP orTBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J.Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res.25:3389-402, each herein incorporated by reference.

pH-Dependent Binding

Provided herein are anti-PRLR antibodies with pH-dependent bindingcharacteristics. For example, an anti-PRLR antibody as described hereinmay exhibit reduced binding to PRLR at acidic pH as compared to neutralpH. Alternatively, anti-PRLR antibodies may exhibit enhanced binding toPRLR at acidic pH as compared to neutral pH. The expression “acidic pH”includes pH values less than about 6.2, e.g., about 6.0, 5.95, 5.9,5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25,5.2, 5.15, 5.1, 5.05, 5.0, or less. As used herein, the expression“neutral pH” means a pH of about 7.0 to about 7.4. The expression“neutral pH” includes pH values of about 7.0, 7.05, 7.1, 7.15, 7.2,7.25, 7.3, 7.35, and 7.4.

In certain instances, “reduced binding to PRLR at acidic pH as comparedto neutral pH” is expressed in terms of a ratio of the K_(D) value ofthe antibody binding to PRLR at acidic pH to the K_(D) value of theantibody binding to PRLR at neutral pH (or vice versa). For example, anantibody or antigen-binding fragment thereof may be regarded asexhibiting “reduced binding to PRLR at acidic pH as compared to neutralpH” for purposes of the present disclosure if the antibody orantigen-binding fragment thereof exhibits an acidic/neutral K_(D) ratioof about 3.0 or greater. In certain exemplary embodiments, theacidic/neutral K_(D) ratio for an antibody or antigen-binding fragmentof the present disclosure can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5,6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0,12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0,70.0, 100.0 or greater.

Antibodies with pH-dependent binding characteristics may be obtained,e.g., by screening a population of antibodies for reduced (or enhanced)binding to a particular antigen at acidic pH as compared to neutral pH.Additionally, modifications of the antigen-binding domain at the aminoacid level may yield antibodies with pH-dependent characteristics. Forexample, by substituting one or more amino acids of an antigen-bindingdomain (e.g., within a CDR) with a histidine residue, an antibody withreduced antigen-binding at acidic pH relative to neutral pH may beobtained.

Anti-PRLR Antibodies Comprising Fc Variants

According to certain embodiments of the present disclosure, anti-PRLRantibodies are provided comprising an Fc domain comprising one or moremutations which enhance or diminish antibody binding to the FcRnreceptor, e.g., at acidic pH as compared to neutral pH. For example, theantibodies useful herein are anti-PRLR antibodies comprising a mutationin the C_(H)2 or a C_(H)3 region of the Fc domain, wherein themutation(s) increases the affinity of the Fc domain to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0). Such mutations may result in an increase in serum half-lifeof the antibody when administered to an animal. Non-limiting examples ofsuch Fc modifications include, e.g., a modification at position 250(e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T),254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification atposition 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., H/For Y); or a modification at position 250 and/or 428; or a modificationat position 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment,the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S)modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F)modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification;a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Qand 428L modification (e.g., T250Q and M428L); and a 307 and/or 308modification (e.g., 308F or 308P).

For example, the antibodies useful herein include anti-PRLR antibodiescomprising an Fc domain comprising one or more pairs or groups ofmutations selected from the group consisting of: 250Q and 248L (e.g.,T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E);428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433Kand N434F). All possible combinations of the foregoing Fc domainmutations, and other mutations within the antibody variable domainsdisclosed herein, are contemplated within the scope of the presentdisclosure.

Biological Characteristics of the Antibodies

Useful in the methods provided herein are ADCs comprising antibodies andantigen-binding fragments thereof that bind monomeric human PRLR withhigh affinity. For example, these antibodies include anti-PRLRantibodies that bind monomeric human PRLR (e.g., hPRLR.mmh) with a K_(D)of less than about 4.0 nM as measured by surface plasmon resonance at25° C. or 37° C., e.g., using an assay format as defined in Example 3herein, or a substantially similar assay. According to certainembodiments, anti-PRLR antibodies are provided that bind monomeric humanPRLR at 37° C. with a K_(D) of less than about 4 nM, less than about 3nM, less than about 2 nM, less than about 1 nM, less than about 900 pM,less than about 800 pM, less than about 700 pM, less than about 600 pM,less than about 500 pM, less than about 400 pM, or less than about 300pM, as measured by surface plasmon resonance, e.g., using an assayformat as defined in Example 3 herein, or a substantially similar assay.

Also useful herein are antibodies and antigen-binding fragments thereofthat bind monomeric human PRLR (e.g., hPRLR.mmh) with a dissociativehalf-life (t½) of greater than about 5 minutes as measured by surfaceplasmon resonance at 25° C. or 37° C., e.g., using an assay format asdefined in Example 3 herein, or a substantially similar assay. Accordingto certain embodiments, anti-PRLR antibodies are provided that bindmonomeric human PRLR at 37° C. with a t½ of greater than about 5minutes, greater than about 6 minutes, greater than about 8 minutes,greater than about 10 minutes, greater than about 12 minutes, greaterthan about 14 minutes, greater than about 16 minutes, greater than about18 minutes, greater than about 20 minutes, greater than about 30minutes, greater than about 40 minutes, or longer, as measured bysurface plasmon resonance, e.g., using an assay format as defined inExample 3 herein, or a substantially similar assay.

Also useful herein are antibodies and antigen-binding fragments thereofthat bind dimeric human PRLR (e.g., hPRLR.mFc) with high affinity. Forexample, such anti-PRLR antibodies can bind dimeric human PRLR with aK_(D) of less than about 250 pM as measured by surface plasmon resonanceat 25° C. or 37° C., e.g., using an assay format as defined in Example 3herein, or a substantially similar assay. According to certainembodiments, anti-PRLR antibodies are provided that bind dimeric humanPRLR at 37° C. with a K_(D) of less than about 250 pM, less than about200 pM, less than about 180 pM, less than about 160 pM, less than about140 pM, less than about 120 pM, less than about 100 pM, less than about80 pM, less than about 70 pM, or less than about 60 pM, as measured bysurface plasmon resonance, e.g., using an assay format as defined inExample 3 herein, or a substantially similar assay.

Also useful herein are antibodies and antigen-binding fragments thereofthat bind dimeric human PRLR (e.g., hPRLR.mFc) with a dissociativehalf-life (t½) of greater than about 55 minutes as measured by surfaceplasmon resonance at 25° C. or 37° C., e.g., using an assay format asdefined in Example 3 herein, or a substantially similar assay. Accordingto certain embodiments, anti-PRLR antibodies are provided that binddimeric human PRLR at 37° C. with a t½ of greater than about 55 minutes,greater than about 60 minutes, greater than about 65 minutes, greaterthan about 70 minutes, greater than about 75 minutes, greater than about80 minutes, greater than about 85 minutes, greater than about 90minutes, greater than about 95 minutes, greater than about 100 minutes,greater than about 120 minutes, greater than about 140 minutes, greaterthan about 160 minutes, or longer, as measured by surface plasmonresonance, e.g., using an assay format as defined in Example 3 herein,or a substantially similar assay.

Also useful herein are antibodies and antigen-binding fragments thereofthat bind PRLR and block prolactin-mediated signaling in cellsexpressing human PRLR. For example, such antibodies include anti-PRLRantibodies that block prolactin-mediated signaling in cells that expresshuman PRLR, with an IC₅₀ of less than about 1.3 nM as measured using aprolactin signaling blocking assay, e.g., using an assay format asdefined in Example 5 herein, or a substantially similar assay. Accordingto certain embodiments, anti-PRLR antibodies are provided that blockprolactin-mediated signaling in cells expressing human PRLR, with anIC₅₀ of less than about 1.3 nM, less than about 1.2 nM, less than about1.0 nM, less than about 900 pM, less than about 800 pM, less than about600 pM, less than about 400 pM, less than about 200 pM, less than about100 pM, less than about 80 pM, less than about 60 pM, less than about 40pM, less than about 20 pM as measured using a prolactin signalingblocking assay, e.g., using an assay format as defined in Example 5herein, or a substantially similar assay.

Also useful herein are antibodies and antigen-binding fragments thereofthat bind PRLR but do not block prolactin-mediated signaling in cellsexpressing human PRLR. As used herein, an antibody or antigen-bindingfragment thereof “does not block” prolactin-mediated signaling if, whentested in a prolactin signaling blocking assay such as the assay definedin Example 5 herein or a substantially similar assay, the antibodyexhibits no or only negligible blocking activity. According to certainembodiments, an antibody or antigen-binding fragment “does not block”prolactin-mediated signaling if the antibody exhibits an IC₅₀ value ofgreater than about 10 nM, or greater than about 100 nM when tested in aprolactin signaling blocking assay such as the assay defined in Example5 herein or a substantially similar assay.

The antibodies useful herein may possess one or more of theaforementioned biological characteristics, or any combination thereof.The foregoing list of biological characteristics of the antibodies ofthe disclosure is not intended to be exhaustive. Other biologicalcharacteristics of the antibodies of the present disclosure will beevident to a person of ordinary skill in the art from a review of thepresent disclosure including the working Examples herein.

Antibody-Drug Conjugates (ADCs)

Useful according to the methods provided herein are antibody-drugconjugates (ADCs) comprising an anti-PRLR antibody or antigen-bindingfragment thereof conjugated to a therapeutic moiety such as a cytotoxicagent, a chemotherapeutic drug, or a radioisotope.

Cytotoxic agents include any agent that is detrimental to the growth,viability or propagation of cells. Examples of suitable cytotoxic agentsand chemotherapeutic agents that can be conjugated to anti-PRLRantibodies in accordance with this aspect of the disclosure include,e.g., 1-(2chloroethyl)-1,2-dimethanesulfonyl hydrazide,1,8-dihydroxy-bicyclo[7.3.1]trideca-4,9-diene-2,6-diyne-13-one,1-dehydrotestosterone, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine,9-amino camptothecin, actinomycin D, amanitins, aminopterin, anguidine,anthracycline, anthramycin (AMC), auristatins, bleomycin, busulfan,butyric acid, calicheamicins, camptothecin, carminomycins, carmustine,cemadotins, cisplatin, colchicin, combretastatins, cyclophosphamide,cytarabine, cytochalasin B, dactinomycin, daunorubicin, decarbazine,diacetoxypentyldoxorubicin, dibromomannitol, dihydroxy anthracin dione,disorazoles, dolastatin (e.g., dolastatin 10), doxorubicin, duocarmycin,echinomycins, eleutherobins, emetine, epothilones, esperamicin,estramustines, ethidium bromide, etoposide, fluorouracils,geldanamycins, gramicidin D, glucocorticoids, irinotecans, kinesinspindle protein (KSP) inhibitors, leptomycins, leurosines, lidocaine,lomustine (CCNU), maytansinoids, mechlorethamine, melphalan,mercatopurines, methopterins, methotrexate, mithramycin, mitomycin,mitoxantrone, N8-acetyl spermidine, podophyllotoxins, procaine,propranolol, pteridines, puromycin, pyrrolobenzodiazepines (PBDs),rhizoxins, streptozotocin, tallysomycins, taxol, tenoposide, tetracaine,thioepa chlorambucil, tomaymycins, topotecans, tubulysin, vinblastine,vincristine, vindesine, vinorelbines, and derivatives of any of theforegoing. Additional cytotoxic agents include a tomaymycin derivativeor a dolastatin derivative. According to certain embodiments, thecytotoxic agent that is conjugated to an anti-PRLR antibody is anauristatin such as MMAE, MMAF, or derivatives thereof. Other cytotoxicagents known in the art are contemplated within the scope of the presentdisclosure, including, e.g., protein toxins such ricin, C. difficiletoxin, pseudomonas exotoxin, ricin, diphtheria toxin, botulinum toxin,bryodin, saporin, pokeweed toxins (i.e., phytolaccatoxin andphytolaccigenin), and others such as those set forth in Sapra et al.,Pharmacol. & Therapeutics, 2013, 138:452-469.

Maytansinoids

According to certain embodiments, the cytotoxic agent that is conjugatedto an anti-PRLR antibody is a maytansinoid. Maytansinoids are analogs ofmaytansine, a potent microtubule targeted compound that induces mitoticarrest. Suitable maytansinoids for the conjugates described hereininclude, but are not limited to DM1 and DM4. Suitable maytansinoids alsoinclude, but are not limited to, those having the structure of Formula(I):

wherein A is arylene or heteroarylene.

Suitable maytansinoids also include, but are not limited to, thosehaving the structure of Formula (II):

wherein:

-   -   A_(3a) is an amino acid, a peptide having 2-20 amino acids, an        alkyl, an alkynyl, an alkenyl, a cycloalkyl, an aryl, a        heteroaryl, a heterocyclyl, —CR₅R₆—, —O—, —C(═O)—, —O—C(═O)—,        —C(═O)—O—, —O—C(═O)—O—, —C(═O)—(CH_(x))_(p1)—,        —C(═O)—O—(CH_(x))_(p1)—, —(CH_(x))_(p1)—C(═O)—,        —(CH_(x))_(p1)—C(═O)—O—, —(O—(CH₂)_(p2)—)_(p3)—,        —((CH₂)_(p2)—O—)_(p3)—, —C(═S)—, —C(═S)—S—, —C(═S)—NH—,        —S—C(═S)—, —S—C(═S)—S—, —S—, —SO—, —SO₂—, —NR₄—,        —N(R₄)—C(═O)—N(R₈)—, —N(R₄)—C(═O)O—, —N(R₄)—C(═O)—,        —C(═O)—N(R₄)—, —C(═O)—N(R₄)—C(═O)—, or —O—C(═O)—NR₄—, wherein        alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, and        heterocyclyl are optionally substituted; and    -   p1, p2 and p3 are each independently 0, or an integer from 1 to        100;    -   x is 0, 1 or 2;    -   R₄, R₅, R₆ and R₈ are each independently H, or a substituted or        unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl, or        heterocyclyl; and    -   R_(4a) is a substituted or unsubstituted: alkyl, alkenyl,        alkynyl, aryl, heteroaryl, or heterocyclyl.

Other suitable maytansinoids include those provided in WO 2014/145090,WO 2016/160615, and WO 2015/031396, incorporated by reference herein intheir entireties.

In some embodiments, the ADC useful in the methods provided hereincomprises the formula M-[L-D]n, wherein M is an antibody orantigen-binding fragment thereof that binds to human prolactin receptor(PRLR), L is a linker, D is a cytotoxic agent, and n is an integer from1 to 20. In certain embodiments, L is4-(N-maleimidomethyl)cyclohexane-1-carboxylate (MCC), and D is DM1 Incertain embodiments, n is an integer from 2 to 5. In certainembodiments, n is 3 or 4.

Radionuclide Conjugates

Also provided herein are antibody-radionuclide conjugates (ARCs)comprising anti-PRLR antibodies conjugated to one or more radionuclides,for example, the chelating moiety. Exemplary radionuclides that can beused in the context of this aspect of the disclosure include, but arenot limited to, e.g., ²²⁵Ac, ²¹²Bi, ²¹³Bi, ¹³¹I, ¹⁸⁶Re, ²²⁷Th, ²²²Rn,²²³Ra, ²²⁴Ra, and ⁹⁰Y.

Linkers

In certain embodiments, ADCs useful herein comprise an anti-PRLRconjugated to a cytotoxic agent (e.g., any of the cytotoxic agentsdisclosed above) via a linker molecule. As used herein, the phrase“linker” refers to any divalent group or moiety that links, connects, orbonds an antibody, or an antigen-binding fragment thereof, with apayload compound set forth herein (e.g., cytotoxic agent). Generally,suitable linkers for the antibody conjugates described herein are thosethat are sufficiently stable to exploit the circulating half-life of theantibody and, at the same time, capable of releasing its payload afterantigen-mediated internalization of the conjugate. Linkers can becleavable or non-cleavable. Cleavable linkers are linkers that arecleaved by intracellular metabolism following internalization, e.g.,cleavage via hydrolysis, reduction, or enzymatic reaction. Non-cleavablelinkers are linkers that release an attached payload via lysosomaldegradation of the antibody following internalization.

Suitable linkers include, but are not limited to, acid-labile linkers,hydrolysis-labile linkers, enzymatically cleavable linkers, cathepsincleavable linkers, reduction labile linkers, self-immolative linkers,and β-gluconuride linkers. Suitable linkers also include, but are notlimited to, those that are or comprise glucuronides,succinimide-thioethers, polyethylene glycol (PEG) units, hydrazones,mal-caproyl (mc) units, disulfide units (e.g., —S—S—, —S—C(R¹R²)—,wherein R¹ and R² are independently hydrogen or hydrocarbyl), carbamateunits, para-amino-benzyl units (PAB; e.g., derived from p-amino benzylalcohol), phosphate units, e.g., mono-, bis-, or tris-phosphate units,and peptide units, e.g., peptide units containing two, three four, five,six, seven, eight, or more amino acids, including but not limited toVal-Cit and Phe-Lys units. Suitable linkers also include MCC(4-[-maleimidomethyl] cyclohexane-1-carboxylate), SPDB, mc-val-cit, andmc-val-cit-PAB.

Linkers can be bonded to the antibody or antigen-binding fragmentthereof through any suitable amino acid of the antibody orantigen-binding fragment. Such amino acids include, but are not limitedto lysine, cysteine, selenocysteine, non-natural amino acids, and acidicamino acids. Linkers can also be conjugated to glutamine viatransglutaminase-based chemo-enzymatic conjugation (see, e.g., Dennleret al., Bioconjugate Chem. 2014, 25, 569-578). Linkers can be coupled tocysteine residues, for example, by reacting the antibody with a linkercontaining a suitable reactive moiety, e.g., a maleimido group. Linkerscan be coupled to lysine residues, for example, by reacting the antibodywith a linker containing an activated ester or acid halide group.

Antibodies can also be conjugated via click chemistry reaction. In someembodiments of said click chemistry reaction, the linker comprises areactive group, e.g., alkyne that is capable of undergoing a 1,3cycloaddition reaction with an azide. Such suitable reactive groupsinclude, but are not limited to, strained alkynes, e.g., those suitablefor strain-promoted alkyne-azide cycloadditions (SPAAC), cycloalkynes,e.g., cyclooctynes, benzannulated alkynes, and alkynes capable ofundergoing 1,3 cycloaddition reactions with azides in the absence ofcopper catalysts. Suitable alkynes also include, but are not limited to,DIBAC, DIBO, BARAC, DIFO, substituted, e.g., fluorinated alkynes,aza-cycloalkynes, BCN, and derivatives thereof. Linkers comprising suchreactive groups are useful for conjugating antibodies that have beenfunctionalized with azido groups. Such functionalized antibodies includeantibodies functionalized with azido-polyethylene glycol groups. Incertain embodiments, such functionalized antibody is derived by reactingan antibody comprising at least one glutamine residue, e.g., heavy chainQ295, with a compound according to the formula H₂N-LL-N₃, wherein LL isa divalent polyethylene glycol group, in the presence of the enzymetransglutaminase.

Any method known in the art for conjugating a chemical moiety to apeptide, polypeptide or other macromolecule can be used in the contextof the present disclosure to make an anti-PRLR ADC as described herein.An exemplary method for antibody-drug conjugation via a linker is setforth in Example 6 herein. Variations on this exemplary method will beappreciated by persons of ordinary skill in the art and are contemplatedwithin the scope of the present disclosure.

Targeting ADCs to Cells Expressing Low Levels of PRLR

It was surprisingly discovered by the present inventors that ADCscomprising an anti-PRLR antibody conjugated to a cytotoxic agent areable to specifically target and kill cells that express relatively lowlevels of cell surface PRLR. For example, in Example 7 herein, it isshown that an ADC comprising anti-PRLR antibody H1H6953N conjugated toDM1 was able to inhibit the growth of T47D cells (expressing PRLR atonly 12× above background) with an IC₅₀ of 1.3 nM and showed 78%killing. By contrast, ADCs against other tumor-associated antigens suchas ErbB2 typically require much higher expression levels of the targetantigen on cells for comparable killing potencies. For example, cellkilling in the sub-nanomolar IC₅₀ range was obtained with ananti-ErbB2-DM1 ADC only with cells that express ErbB2 at levels ofgreater than about 200× to about 400× above background (see, e.g.,Tables 14-17 herein). The ability to kill tumor cells that expressrelatively low levels of tumor-associated antigen such as PRLR meansthat the anti-PRLR ADCs of the present disclosure can providesignificant therapeutic benefits with a lower dose and/or less frequentdosing than is required for ADCs that target other tumor antigens suchas ErbB2.

Accordingly, the present disclosure provides antibody-drug conjugates(ADCs) comprising an antibody or antigen-binding fragment thereof thatspecifically binds human PRLR conjugated to a cytotoxic agent, whereinthe ADCs effectively kill cells (e.g., tumor cells) that express lowlevels of PRLR. In related embodiments, the present disclosure includesmethods for effectively killing cells that express low levels of PRLR.The methods according to this aspect of the disclosure comprise (1)contacting the cells with an antibody-drug conjugate (ADC) comprising ananti-PRLR antibody conjugated to a cytotoxic agent and (2) contactingthe cells with fulvestrant. “Contacting the cells” can be carried out invitro, or in vivo, e.g., by administering an anti-PRLR ADC to a subjectin need thereof, wherein the administration causes the ADC andfulvestrant to come into contact with cells expressing PRLR.

According to certain contexts envisioned within the scope of the presentdisclosure, a “low level of PRLR” means an expression level of less thanabout 30-fold above background. According to certain embodiments,anti-PRLR ADCs are provided which effectively kill cells that expressPRLR at an expression level of less than about 30-fold, 25-fold,20-fold, 18-fold, 16-fold, 14-fold, 12-fold, 10-fold, 8-fold, or less,above background. As used herein, the term “background” means the(non-specific) signal produced when cells are treated with an isotypecontrol antibody (i.e., not specific for PRLR).

In certain other contexts, a “low level of PRLR” can be expressed interms of the number of PRLR molecules per cell. For example, as usedherein, a cell that expresses a “low level” of PRLR expresses less thanabout 1 million copies of PRLR per cell. In specific embodiments, a “lowlevel” of PRLR means less than about 900,000 copies, less than about800,000 copies, less than about 700,000 copies, less than about 600,000copies, less than about 500,000 copies, less than about 400,000 copies,less than about 300,000 copies, less than about 200,000 copies, lessthan about 100,000 copies, less than about 90,000 copies, less thanabout 80,000 copies, less than about 70,000 copies, less than about60,000 copies, less than about 50,000 copies, less than about 40,000copies, less than about 30,000 copies, less than about 20,000 copies,less than about 10,000 copies of PRLR per cell, less than about 5,000copies of PRLR per cell, less than about 3,000 copies of PRLR per cell,or less than about 1,000 copies of PRLR per cell. For example, as shownin the working examples herein, anti-PRLR ADCs of the present disclosurewere shown to effectively kill MCF7 cells, which only expressapproximately 3000 copies of PRLR per cell.

As used herein, “effective killing” means that the ADC exhibits an IC₅₀of less than about 20 nM, or less than about 1 nM (e.g., less than about0.9 nM, less than about 0.8 nM, less than about 0.7 nM, less than about0.6 nM, less than about 0.5 nM, less than about 0.4 nM, or less thanabout 0.3 nM) in a tumor cell killing assay, such as the assay definedin Example 7 herein, or a substantially similar assay. According to thisaspect of the disclosure, the anti-PRLR antibody component of the ADCcan be any anti-PRLR antibody including anti-PRLR antibodies comprisingany of the CDR or HCVR/LCVR amino acid sequences as set forth in Table 1herein. Additionally, the cytotoxic agent component of the ADC can beany cytotoxic agent, such as DM1, or any other cytotoxic agent mentionedherein.

ADCs useful in the methods of the present disclosure are able to inhibittumor growth and/or reduce tumor size in PRLR+ tumor-bearing animals.For example, as shown in Example 8 herein, anti-PRLR-DM1 ADCs were shownto reduce tumors to undetectable levels in mice bearing PRLR+ breastcancer xenografts. Thus, provided herein are methods of using anti-PRLRantibodies and ADCs comprising such antibodies, wherein the antibodiesor ADCs, when administered in combination with fulvestrant to a PRLR+tumor-bearing animal (e.g., at a frequency of about once a week, and adose of about 1 to 15 mg/kg), inhibit tumor growth and/or reduce tumorsize (e.g., tumor growth inhibition of 100% or greater) by Day 52post-administration or sooner.

Class I Cytokine Receptor Targeting

PRLR belongs to the class I cytokine receptor family. As explained aboveand demonstrated in the working examples herein, it was unexpectedlydiscovered that antibody-drug conjugates (ADCs) comprising anti-PRLRantibodies can effectively target and kill cells that express low levelsof PRLR (see Example 7 herein). Furthermore, it was shown that ADCsagainst other class I cytokine receptors (IL-4R and IL-6R) also are ableto potently kill cell lines expressing relatively low levels of targetantigen (see Example 9 herein). This property is in contrast to ADCsagainst other cell surface-expressed proteins, such as ErbB2, whereineffective cell killing requires high target expression. Moreover, it wasalso surprisingly discovered that anti-PRLR antibodies are internalizedsubstantially faster than anti-Her2 antibodies on tumor cells (e.g.,T47D tumor cells), and that this property is correlated with fasterinternalization and degradation of cell surface PRLR compared to cellsurface Her2.

It was also surprisingly discovered that treatment of PRLR positivetumor cells with the selective estrogen receptor down-regulator (SERD),fulvestrant, did not decrease PRLR expression. This is in contrast toprevious findings that treatment with tamoxifen, which blocks binding ofestrogen to the estrogen receptor, caused a decline in PRLR mRNA sevendays post treatment. Swaminathan et al., 2009, J. Mammary Gland Biol.Neoplasia, 13(1): 81-91.

In view of the results set forth herein, the present inventors conceivedthat the ability to target and kill cells that express low levels ofcell surface antigen may be a common property shared by ADCs directedagainst class I cytokine receptors in general, and in particular class Icytokine receptors that are rapidly internalized. Thus, the presentdisclosure includes methods for targeting class I cytokine receptors(e.g., rapidly internalizing class I cytokine receptors), and methodsfor killing cells that express class I cytokine receptors such as cellsthat express low levels of class I cytokine receptors.

The methods according to this aspect of the disclosure comprisecontacting a cell that expresses a class I cytokine receptor with an ADCcomprising an antibody or antigen-binding fragment thereof thatspecifically binds the class I cytokine receptor. According to certainembodiments, the cell to be targeted expresses low levels (as thatexpression is defined elsewhere herein) of a class I cytokine receptorand/or a class I cytokine receptor that is rapidly internalized anddegraded (e.g., internalized faster than a reference cell surfacemolecule such as Her2). Also included within the present disclosure areADCs comprising an antibody or antigen-binding fragment thereof thatspecifically binds a class I cytokine receptor, conjugated to acytotoxic agent. Any of the cytotoxic agents, linkers, and/orADC-related technologies described elsewhere herein can be used in thecontext of this aspect of the disclosure.

As used herein a “class I cytokine receptor” (also sometimes referred toas a “type I cytokine receptor”) means a transmembrane receptorexpressed on the surface of cells that recognizes and responds tocytokines with four alpha-helical strands. As explained below, class Icytokine receptors can be heterodimeric or homodimeric. As used herein,the term “class I cytokine receptor” includes both heterodimeric andhomodimeric receptors.

Heterodimeric class I cytokine receptors consist of a cytokine-specificchain and a “common chain.” Accordingly, such heterodimeric class Icytokine receptors can be classified based on the type of common chainused by the receptor for signaling. Exemplary categories ofheterodimeric class I cytokine receptors include: (i) common gammachain-containing heterodimeric receptors such as IL-2R, IL-4R, IL-7R,IL-9R, IL-13R and IL-15R; (ii) common beta chain-containingheterodimeric receptors such as GM-CSF receptor, IL-3R and IL-5R; and(iii) gp130-containing heterodimeric receptors such as IL-6R, IL-11R,CNTF receptor, leukemia inhibitory factor (LIF) receptor, oncostatin M(OSM) receptor, and IL-12 receptor.

Homodimeric class I cytokine receptors include growth hormone (GH)receptor, erythropoietin (EPO) receptor, G-CSF receptor, leptinreceptor, and PRLR.

In certain embodiments of this aspect of the disclosure, the ADCcomprises an antibody or antigen-binding fragment thereof thatspecifically binds a heterodimeric class I cytokine receptor. Accordingto other embodiments of this aspect of the disclosure, the ADC comprisesan antibody or antigen-binding fragment thereof that specifically bindsa homodimeric class I cytokine receptor.

The present disclosure includes methods for killing a cell thatexpresses low levels of a heterodimeric class I cytokine receptor. Themethods according to this aspect of the disclosure comprise contacting acell that expresses a low level of a heterodimeric class I cytokinereceptor with an ADC comprising an antibody or antigen-binding fragmentthereof that specifically binds the heterodimeric class I cytokinereceptor.

Alternatively, the present disclosure includes methods for killing acell that expresses low levels of a homodimeric class I cytokinereceptor. The methods according to this aspect of the disclosurecomprise contacting a cell that expresses a low level of a homodimericclass I cytokine receptor with an ADC comprising an antibody orantigen-binding fragment thereof that specifically binds the homodimericclass I cytokine receptor.

Epitope Mapping and Related Technologies

The epitope to which the antibodies of the present disclosure bind mayconsist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acidsof a PRLR protein. Alternatively, the epitope may consist of a pluralityof non-contiguous amino acids (or amino acid sequences) of PRLR. In someembodiments, the epitope is located on or near the prolactin-bindingdomain of PRLR. In other embodiments, the epitope is located outside ofthe prolactin-binding domain of PRLR, e.g., at a location on the surfaceof PRLR at which an antibody, when bound to such an epitope, does notinterfere with prolactin binding to PRLR.

The PRLR extracellular domain consists of two fibronectin-like type IIIdomains, referred to herein as “Domain 1” and “Domain 2.” Domain 1 isthe sequence of amino acids represented by amino acids 27 through 128 ofSEQ ID NO:404; and Domain 2 is the sequence of amino acids representedby amino acids 129 through 229 of SEQ ID NO:404. The present disclosureincludes anti-PRLR antibodies which interact with one or more epitopesfound within Domain 1 of the extracellular domain of PRLR. Theepitope(s) may consist of one or more contiguous sequences of 3 or more(e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20or more) amino acids located within Domain 1 of PRLR. Alternatively, theepitope may consist of a plurality of non-contiguous amino acids (oramino acid sequences) located within Domain 1 of PRLR. As shown inExample 10 herein, the epitope of PRLR with which the exemplary antibodyof the disclosure HIH6958N2 interacts is defined by the amino acidsequence MHECPDYITGGPNSCHFGKQYTSMWRTYIMM (SEQ ID NO:405), whichcorresponds to amino acids 72-102 of SEQ ID NO:404. Accordingly, thepresent disclosure includes anti-PRLR antibodies that interact with oneor more amino acids contained within the Domain 1 amino acid segmentconsisting of amino acids 72-102 of SEQ ID NO:404 (i.e., the sequenceMHECPDYITGGPNSCHFGKQYTSMWRTYIMM [SEQ ID NO:405]).

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antibody “interacts with one or more aminoacids” within a polypeptide or protein. Exemplary techniques include,e.g., routine cross-blocking assay such as that described Antibodies,Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., N.Y.),alanine scanning mutational analysis, peptide blots analysis (Reineke,2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. Inaddition, methods such as epitope excision, epitope extraction andchemical modification of antigens can be employed (Tomer, 2000, ProteinScience 9:487-496). Another method that can be used to identify theamino acids within a polypeptide with which an antibody interacts ishydrogen/deuterium exchange detected by mass spectrometry. In generalterms, the hydrogen/deuterium exchange method involvesdeuterium-labeling the protein of interest, followed by binding theantibody to the deuterium-labeled protein. Next, the protein/antibodycomplex is transferred to water to allow hydrogen-deuterium exchange tooccur at all residues except for the residues protected by the antibody(which remain deuterium-labeled). After dissociation of the antibody,the target protein is subjected to protease cleavage and massspectrometry analysis, thereby revealing the deuterium-labeled residueswhich correspond to the specific amino acids with which the antibodyinteracts. See, e.g., Ehring (1999) Analytical Biochemistry267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

Provided herein are anti-PRLR antibodies that bind to the same epitopeas any of the specific exemplary antibodies described herein (e.g.antibodies comprising any of the amino acid sequences as set forth inTable 1 herein). Likewise, anti-PRLR antibodies that compete for bindingto PRLR with any of the specific exemplary antibodies described hereinare also provided (e.g. antibodies comprising any of the amino acidsequences as set forth in Table 1 herein).

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-PRLR antibody byusing routine methods known in the art and exemplified herein. Forexample, to determine if a test antibody binds to the same epitope as areference anti-PRLR antibody of the disclosure, the reference antibodyis allowed to bind to a PRLR protein. Next, the ability of a testantibody to bind to the PRLR molecule is assessed. If the test antibodyis able to bind to PRLR following saturation binding with the referenceanti-PRLR antibody, it can be concluded that the test antibody binds toa different epitope than the reference anti-PRLR antibody. On the otherhand, if the test antibody is not able to bind to the PRLR moleculefollowing saturation binding with the reference anti-PRLR antibody, thenthe test antibody may bind to the same epitope as the epitope bound bythe reference anti-PRLR antibody of the disclosure. Additional routineexperimentation (e.g., peptide mutation and binding analyses) can thenbe carried out to confirm whether the observed lack of binding of thetest antibody is in fact due to binding to the same epitope as thereference antibody or if steric blocking (or another phenomenon) isresponsible for the lack of observed binding. Experiments of this sortcan be performed using ELISA, RIA, Biacore, flow cytometry or any otherquantitative or qualitative antibody-binding assay available in the art.In accordance with certain embodiments of the present disclosure, twoantibodies bind to the same (or overlapping) epitope if, e.g., a 1-, 5-,10-, 20- or 100-fold excess of one antibody inhibits binding of theother by at least 50% but preferably 75%, 90% or even 99% as measured ina competitive binding assay (see, e.g., Junghans et al., Cancer Res.1990:50:1495-1502). Alternatively, two antibodies are deemed to bind tothe same epitope if essentially all amino acid mutations in the antigenthat reduce or eliminate binding of one antibody reduce or eliminatebinding of the other. Two antibodies are deemed to have “overlappingepitopes” if only a subset of the amino acid mutations that reduce oreliminate binding of one antibody reduce or eliminate binding of theother.

To determine if an antibody competes for binding (or cross-competes forbinding) with a reference anti-PRLR antibody, the above-describedbinding methodology is performed in two orientations: In a firstorientation, the reference antibody is allowed to bind to a PRLR proteinunder saturating conditions followed by assessment of binding of thetest antibody to the PRLR molecule. In a second orientation, the testantibody is allowed to bind to a PRLR molecule under saturatingconditions followed by assessment of binding of the reference antibodyto the PRLR molecule. If, in both orientations, only the first(saturating) antibody is capable of binding to the PRLR molecule, thenit is concluded that the test antibody and the reference antibodycompete for binding to PRLR. As will be appreciated by a person ofordinary skill in the art, an antibody that competes for binding with areference antibody may not necessarily bind to the same epitope as thereference antibody, but may sterically block binding of the referenceantibody by binding an overlapping or adjacent epitope.

Preparation of Human Antibodies

The anti-PRLR antibodies of the present disclosure can be fully humanantibodies. Methods for generating monoclonal antibodies, includingfully human monoclonal antibodies are known in the art. Any such knownmethods can be used in the context of the present disclosure to makehuman antibodies that specifically bind to human PRLR.

Using VELOCIMMUNE™ technology, for example, or any other similar knownmethod for generating fully human monoclonal antibodies, high affinitychimeric antibodies to PRLR are initially isolated having a humanvariable region and a mouse constant region. As in the experimentalsection below, the antibodies are characterized and selected fordesirable characteristics, including affinity, ligand blocking activity,selectivity, epitope, etc. If necessary, mouse constant regions arereplaced with a desired human constant region, for example wild-type ormodified IgG1 or IgG4, to generate a fully human anti-PRLR antibody.While the constant region selected may vary according to specific use,high affinity antigen-binding and target specificity characteristicsreside in the variable region. In certain instances, fully humananti-PRLR antibodies are isolated directly from antigen-positive Bcells.

Bioequivalents

The anti-PRLR antibodies and antibody fragments described hereinencompass proteins having amino acid sequences that vary from those ofthe described antibodies but that retain the ability to bind human PRLR.Such variant antibodies and antibody fragments comprise one or moreadditions, deletions, or substitutions of amino acids when compared toparent sequence, but exhibit biological activity that is essentiallyequivalent to that of the described antibodies. Likewise, the anti-PRLRantibody-encoding DNA sequences of the present disclosure encompasssequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to the disclosed sequence,but that encode an anti-PRLR antibody or antibody fragment that isessentially bioequivalent to an anti-PRLR antibody or antibody fragmentof the disclosure. Examples of such variant amino acid and DNA sequencesare discussed above.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single does or multipledose. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of anti-PRLR antibodies of the disclosure may beconstructed by, for example, making various substitutions of residues orsequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation. In other contexts,bioequivalent antibodies may include anti-PRLR antibody variantscomprising amino acid changes which modify the glycosylationcharacteristics of the antibodies, e.g., mutations which eliminate orremove glycosylation.

Species Selectivity and Species Cross-Reactivity

Methods provided herein, according to certain embodiments, utilizeanti-PRLR antibodies that bind to human PRLR but not to PRLR from otherspecies. The present disclosure also includes anti-PRLR antibodies thatbind to human PRLR and to PRLR from one or more non-human species. Forexample, the anti-PRLR antibodies of the disclosure may bind to humanPRLR and may bind or not bind, as the case may be, to one or more ofmouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat,sheep, cow, horse, camel, cynomologous, marmoset, rhesus or chimpanzeePRLR. According to certain exemplary embodiments of the presentdisclosure, anti-PRLR antibodies are provided which specifically bindhuman PRLR and cynomolgus monkey (e.g., Macaca fascicularis) PRLR. Otheranti-PRLR antibodies of the disclosure bind human PRLR but do not bind,or bind only weakly, to cynomolgus monkey PRLR.

Multispecific Antibodies

The antibodies useful in the methods of the present disclosure may bemonospecific or multispecific (e.g., bispecific). Multispecificantibodies may be specific for different epitopes of one targetpolypeptide or may contain antigen-binding domains specific for morethan one target polypeptide. See, e.g., Tutt et al., 1991, J. Immunol.147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. Theanti-PRLR antibodies of the present disclosure can be linked to orco-expressed with another functional molecule, e.g., another peptide orprotein. For example, an antibody or fragment thereof can befunctionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other molecularentities, such as another antibody or antibody fragment to produce abi-specific or a multispecific antibody with a second bindingspecificity.

Also provided herein are bispecific antibodies wherein one arm of animmunoglobulin binds human PRLR, and the other arm of the immunoglobulinis specific for a second antigen. The PRLR-binding arm can comprise anyof the HCVR/LCVR or CDR amino acid sequences as set forth in Table 1herein. In certain embodiments, the PRLR-binding arm binds human PRLRand blocks prolactin binding to PRLR. In other embodiments, thePRLR-binding arm binds human PRLR but does not block prolactin bindingto PRLR.

An exemplary bispecific antibody format that can be used in the contextof the present disclosure involves the use of a first immunoglobulin(Ig) C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first andsecond Ig C_(H)3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bispecific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H)3 domain binds Protein A and the second Ig C_(H)3 domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H)3 may further comprise a Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422Iby EU) in the case of IgG4 antibodies. Variations on the bispecificantibody format described above are contemplated within the scope of thepresent disclosure.

Other exemplary bispecific formats that can be used in the context ofthe present disclosure include, without limitation, e.g., scFv-based ordiabody bispecific formats, IgG-scFv fusions, dual variable domain(DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., commonlight chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1/IgG2, dual acting Fab (DAF)-IgG, andMab² bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11,and references cited therein, for a review of the foregoing formats).Bispecific antibodies can also be constructed using peptide/nucleic acidconjugation, e.g., wherein unnatural amino acids with orthogonalchemical reactivity are used to generate site-specificantibody-oligonucleotide conjugates which then self-assemble intomultimeric complexes with defined composition, valency and geometry.(See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

Therapeutic Formulation and Administration

The disclosure provides pharmaceutical compositions comprising theanti-PRLR antibodies-drug conjugates or antigen-binding fragmentsthereof useful in the present disclosure. The pharmaceuticalcompositions of the disclosure are formulated with suitable carriers,excipients, and other agents that provide improved transfer, delivery,tolerance, and the like. A multitude of appropriate formulations can befound in the formulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as LIPOFECTIN™, Life Technologies, Carlsbad, Calif.), DNAconjugates, anhydrous absorption pastes, oil-in-water and water-in-oilemulsions, emulsions carbowax (polyethylene glycols of various molecularweights), semi-solid gels, and semi-solid mixtures containing carbowax.See also Powell et al. “Compendium of excipients for parenteralformulations” PDA (1998) J Pharm Sci Technol 52:238-311.

The dose of antibody administered to a patient may vary depending uponthe age and the size of the patient, target disease, conditions, routeof administration, and the like. The preferred dose is typicallycalculated according to body weight or body surface area. In an adultpatient, it may be advantageous to intravenously administer the antibodyof the present disclosure normally at a single dose of about 0.01 toabout 20 mg/kg body weight, more preferably about 0.02 to about 7, about0.03 to about 5, or about 0.05 to about 3 mg/kg body weight. Dependingon the severity of the condition, the frequency and the duration of thetreatment can be adjusted. Effective dosages and schedules foradministering anti-PRLR antibodies may be determined empirically; forexample, patient progress can be monitored by periodic assessment, andthe dose adjusted accordingly. Moreover, interspecies scaling of dosagescan be performed using well-known methods in the art (e.g., Mordenti etal., 1991, Pharmaceut. Res. 8:1351).

Various delivery systems are known and can be used to administer thepharmaceutical compositions provided herein, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

A pharmaceutical composition of the present disclosure can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present disclosure. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present disclosure. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present disclosure include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park Ill.), to name only a few.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Fla. In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antibodies

The present disclosure includes methods comprising administering to asubject in need thereof a therapeutic composition comprising anantibody-drug conjugate comprising an anti-PRLR antibody (e.g., an ADCcomprising any of the HCVR/LCVR or CDR sequences as set forth in Table 1herein) and co-administering to the same subject fulvestrant. Thetherapeutic composition can comprise any of the anti-PRLRantibodies-drug conjugates, or antigen-binding fragments thereof, orADCs disclosed herein, and a pharmaceutically acceptable carrier ordiluent.

The ADCs useful according to the methods provided herein can be used,inter alia, for the treatment, prevention and/or amelioration of anydisease or disorder associated with or mediated by PRLR expression oractivity, or treatable by blocking the interaction between PRLR andprolactin or otherwise inhibiting PRLR activity and/or signaling, and/orpromoting receptor internalization and/or decreasing cell surfacereceptor number. For example, the ADCs of the present disclosure areuseful for the treatment of tumors that express PRLR and/or that respondto prolactin-mediated signaling, e.g., breast tumors. The ADCs providedherein may also be used to treat primary and/or metastatic tumorsarising in the brain and meninges, oropharynx, lung and bronchial tree,gastrointestinal tract, male and female reproductive tract, muscle,bone, skin and appendages, connective tissue, spleen, immune system,blood forming cells and bone marrow, liver and urinary tract, andspecial sensory organs such as the eye. In certain embodiments, ADCs areused to treat one or more of the following cancers: renal cellcarcinoma, pancreatic carcinoma, head and neck cancer, prostate cancer,malignant gliomas, osteosarcoma, colorectal cancer, gastric cancer(e.g., gastric cancer with MET amplification), malignant mesothelioma,multiple myeloma, ovarian cancer, small cell lung cancer, non-small celllung cancer, synovial sarcoma, thyroid cancer, breast cancer, ormelanoma. In some embodiments, the cancer is a PRLR positive breastcancer.

The anti-PRLR antibodies of the present disclosure are also useful forthe treatment or prevention of one or more diseases or disordersselected from the group consisting of endometriosis, adenomyosis,non-hormonal female fertility contraception, benign breast disease andmastalgia, lactation inhibition, benign prostate hyperplasia, fibroids,hyper- and normoprolactinemic hair loss, and as part of hormone therapyto inhibit mammary epithelial cell proliferation.

In the context of the methods of treatment described herein, theanti-PRLR antibody may be administered as a monotherapy (i.e., as theonly therapeutic agent) or in combination with one or more additionaltherapeutic agents (examples of which are described elsewhere herein).One particularly useful example is fulvestrant.

The present disclosure includes methods for identifying patients who aretreatable with an ADC of the present disclosure by assaying for highlevels of PRLR expression in one or more tissues of the patient such asa tumor tissue. In a related embodiment, the present disclosure includesmethods for treating cancers characterized by high level expression ofPRLR. For example, the present disclosure includes methods of treatmentcomprising administering an anti-PRLR antibody of the disclosure, or ADCthereof (e.g., any of the anti-PRLR ADCs described elsewhere herein), toa subject with a tumor, wherein the tumor has been identified asexpressing high levels of PRLR. In certain embodiments, the tumor isidentified as expressing high levels of PRLR by immunohistochemistry ofa biopsy sample or other imaging techniques such as, e.g., immuno-PETimaging, etc.

Combination Therapies and Formulations

The present disclosure includes compositions and therapeuticformulations useful in the methods provided herein, where thecompositions and formulations comprise any of the anti-PRLRantibody-drug conjugates described herein in combination with one ormore additional therapeutically active components, and methods oftreatment comprising administering such combinations to subjects in needthereof.

The anti-PRLR antibody-drug conjugates described herein may beco-formulated with and/or administered in combination with atherapeutically effective amount of fulvestrant. Treatment with both theanti-PRLR antibody-drug conjugate and fulvestrant is termed“co-administration”.

The anti-PRLR antibodies of the present disclosure may be co-formulatedwith and/or administered in combination with one or more additionaltherapeutically active component(s) selected from the group consistingof: an EGFR antagonist (e.g., an anti-EGFR antibody [e.g., cetuximab orpanitumumab] or small molecule inhibitor of EGFR [e.g., gefitinib orerlotinib]), an antagonist of another EGFR family member such asHer2/ErbB2, ErbB3 or ErbB4 (e.g., anti-ErbB2 [e.g., trastuzumab or T-DM1{KADCYLA®}], anti-ErbB3 or anti-ErbB4 antibody or small moleculeinhibitor of ErbB2, ErbB3 or ErbB4 activity), an antagonist of EGFRvIII(e.g., an antibody that specifically binds EGFRvIII), a cMET anagonist(e.g., an anti-cMET antibody), an IGF1R antagonist (e.g., an anti-IGF1Rantibody), a B-raf inhibitor (e.g., vemurafenib, sorafenib, GDC-0879,PLX-4720), a PDGFR-α inhibitor (e.g., an anti-PDGFR-α antibody), aPDGFR-β inhibitor (e.g., an anti-PDGFR-β antibody or small moleculekinase inhibitor such as, e.g., imatinib mesylate or sunitinib malate),a PDGF ligand inhibitor (e.g., anti-PDGF-A, -B, -C, or -D antibody,aptamer, siRNA, etc.), a VEGF antagonist (e.g., a VEGF-Trap such asaflibercept, see, e.g., U.S. Pat. No. 7,087,411 (also referred to hereinas a “VEGF-inhibiting fusion protein”), anti-VEGF antibody (e.g.,bevacizumab), a small molecule kinase inhibitor of VEGF receptor (e.g.,sunitinib, sorafenib or pazopanib)), a DLL4 antagonist (e.g., ananti-DLL4 antibody disclosed in US 2009/0142354 such as REGN421), anAng2 antagonist (e.g., an anti-Ang2 antibody disclosed in US2011/0027286 such as H1H685P), a FOLH1 antagonist (e.g., an anti-FOLH1antibody), a STEAP1 or STEAP2 antagonist (e.g., an anti-STEAP1 antibodyor an anti-STEAP2 antibody), a TMPRSS2 antagonist (e.g., an anti-TMPRSS2antibody), a MSLN antagonist (e.g., an anti-MSLN antibody), a CA9antagonist (e.g., an anti-CA9 antibody), a uroplakin antagonist (e.g.,an anti-uroplakin [e.g., anti-UPK3A] antibody), a MUC16 antagonist(e.g., an anti-MUC16 antibody), a Tn antigen antagonist (e.g., ananti-Tn antibody), a CLEC12A antagonist (e.g., an anti-CLEC12Aantibody), a TNFRSF17 antagonist (e.g., an anti-TNFRSF17 antibody), aLGRS antagonist (e.g., an anti-LGRS antibody), a monovalent CD20antagonist (e.g., a monovalent anti-CD20 antibody such as rituximab),etc. Other agents that may be beneficially administered in combinationwith ADCs described herein include, e.g., tamoxifen, aromataseinhibitors, and cytokine inhibitors, including small-molecule cytokineinhibitors and antibodies that bind to cytokines such as IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18,or to their respective receptors.

The compositions and therapeutic formulations comprising any of theanti-PRLR antibody-drug conjugates described herein can be administeredin combination (co-administered) with one or more chemotherapeuticagents. Examples of chemotherapeutic agents include alkylating agentssuch as thiotepa and cyclosphosphamide (Cytoxan™); alkyl sulfonates suchas busulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone;mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinicacid; 2-ethylhydrazide; procarbazine; PSK™; razoxane; sizofiran;spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, e.g.paclitaxel (Taxol™, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddocetaxel (Taxotere™; Aventis Antony, France); chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumanalogs such as cisplatin and carboplatin; vinblastine; platinum;etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

The anti-PRLR antibody-drug conjugates of the disclosure may also beadministered and/or co-formulated in combination with antivirals,antibiotics, analgesics, corticosteroids, steroids, oxygen,antioxidants, COX inhibitors, cardioprotectants, metal chelators,IFN-gamma, and/or NSAIDs.

The additional therapeutically active component(s), e.g., any of theagents listed above or derivatives thereof, may be administered justprior to, concurrent with, or shortly after the administration of ananti-PRLR antibody of the present disclosure; (for purposes of thepresent disclosure, such administration regimens are considered theadministration of an anti-PRLR antibody “in combination with” anadditional therapeutically active component, i.e. “co-administered”).The present disclosure includes pharmaceutical compositions in which ananti-PRLR antibody of the present disclosure is co-formulated with oneor more of the additional therapeutically active component(s) asdescribed elsewhere herein.

Administration Regimens

“Co-administration” as used herein refers to administration of the PRLRantibody-drug conjugate in combination with fulvestrant. According tocertain embodiments of the present disclosure, an anti-PRLRantibody-drug conjugate (or a pharmaceutical composition comprising acombination of an anti-PRLR antibody-drug) and fulvestrant may beadministered to the subject at the same time, or at a different point intime, e.g., on different days separated by a predetermined interval(e.g., hours, days, weeks or months over a defined time course). Themethods according to this aspect of the disclosure comprise sequentiallyadministering to a subject one dose or multiple doses of an anti-PRLRantibody-drug conjugate of the disclosure and one dose or multiple dosesof fulvestrant. In addition, an anti-PRLR antibody-drug conjugate can beadministered prior to or after administration of fulvestrant.

According to certain embodiments of the present disclosure, in additionto administration of fulvestrant, multiple doses of an anti-PRLRantibody-drug conjugate (or a pharmaceutical composition comprising acombination of an anti-PRLR antibody-drug conjugate and any of theadditional therapeutically active agents mentioned herein) may beadministered to a subject over a defined time course. The methodsaccording to this aspect of the disclosure comprise sequentiallyadministering to a subject multiple doses of an anti-PRLR antibody-drugconjugate of the disclosure and/or sequentially administering to thesubject multiple doses of fulvestrant. As used herein, “sequentiallyadministering” means that each dose of anti-PRLR antibody-drug conjugateand/or fulvestrant is administered to the subject at a different pointin time, e.g., on different days separated by a predetermined interval(e.g., hours, days, weeks or months). The present disclosure includesmethods which comprise sequentially administering to the patient asingle initial dose of an anti-PRLR antibody-drug conjugate, followed byone or more secondary doses of the anti-PRLR antibody-drug conjugate,and optionally followed by one or more tertiary doses of the anti-PRLRantibody-drug conjugate. The present disclosure further includes methodswhich comprise sequentially administering to the patient a singleinitial dose of fulvestrant, followed by one or more secondary doses offulvestrant, and optionally followed by one or more tertiary doses offulvestrant.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the therapeutic agentsprovided herein. Thus, the “initial dose” is the dose which isadministered at the beginning of the treatment regimen (also referred toas the “baseline dose”); the “secondary doses” are the doses which areadministered after the initial dose; and the “tertiary doses” are thedoses which are administered after the secondary doses. The initial,secondary, and tertiary doses may all contain the same amount ofanti-PRLR antibody-drug conjugate or same amount of fulvestrant, butgenerally may differ from one another in terms of frequency ofadministration. In certain embodiments, however, the amount of anti-PRLRantibody-drug conjugate contained in the initial, secondary and/ortertiary doses varies from one another (e.g., adjusted up or down asappropriate) during the course of treatment. In certain embodiments, twoor more (e.g., 2, 3, 4, or 5) doses are administered at the beginning ofthe treatment regimen as “loading doses” followed by subsequent dosesthat are administered on a less frequent basis (e.g., “maintenancedoses”).

In certain exemplary embodiments of the present disclosure, eachsecondary and/or tertiary dose is administered 1 to 26 (e.g., 1, 1½, 2,2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½,12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½,20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more)weeks after the immediately preceding dose. The phrase “the immediatelypreceding dose,” as used herein, means, in a sequence of multipleadministrations, the dose of anti-PRLR antibody-drug conjugate, orfulvestrant, which is administered to a patient prior to theadministration of the very next dose in the sequence with no interveningdoses.

The methods according to this aspect of the disclosure may compriseadministering to a patient any number of secondary and/or tertiary dosesof an anti-PRLR antibody-drug conjugate or fulvestrant. For example, incertain embodiments, only a single secondary dose is administered to thepatient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8,or more) secondary doses are administered to the patient. Likewise, incertain embodiments, only a single tertiary dose is administered to thepatient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8,or more) tertiary doses are administered to the patient. Theadministration regimen may be carried out indefinitely over the lifetimeof a particular subject, or until such treatment is no longertherapeutically needed or advantageous.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to2 weeks or 1 to 2 months after the immediately preceding dose.Similarly, in embodiments involving multiple tertiary doses, eachtertiary dose may be administered at the same frequency as the othertertiary doses. For example, each tertiary dose may be administered tothe patient 2 to 12 weeks after the immediately preceding dose. Incertain embodiments of the disclosure, the frequency at which thesecondary and/or tertiary doses are administered to a patient can varyover the course of the treatment regimen. The frequency ofadministration may also be adjusted during the course of treatment by aphysician depending on the needs of the individual patient followingclinical examination.

The present disclosure includes administration regimens in which 2 to 6loading doses are administered to a patient at a first frequency (e.g.,once a week, once every two weeks, once every three weeks, once a month,once every two months, etc.), followed by administration of two or moremaintenance doses to the patient on a less frequent basis. For example,according to this aspect of the disclosure, if the loading doses areadministered at a frequency of once a month, then the maintenance dosesmay be administered to the patient once every six weeks, once every twomonths, once every three months, etc.

While the disclosure has been particularly shown and described withreference to a number of embodiments, it would be understood by thoseskilled in the art that changes in the form and details may be made tothe various embodiments disclosed herein without departing from thespirit and scope of the disclosure and that the various embodimentsdisclosed herein are not intended to act as limitations on the scope ofthe claims.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions provided herein, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1: Generation of Human Anti-PRLR Antibodies

Anti-PRLR antibodies were obtained by immunizing a VELOCIMMUNE® mouse(i.e., an engineered mouse comprising DNA encoding human immunoglobulinheavy and kappa light chain variable regions) with an immunogencomprising a soluble dimeric ecto domain of human PRLR. The antibodyimmune response was monitored by a PRLR-specific immunoassay. When adesired immune response was achieved splenocytes were harvested andfused with mouse myeloma cells to preserve their viability and formhybridoma cell lines. The hybridoma cell lines were screened andselected to identify cell lines that produce PRLR-specific antibodies.Using this technique several anti-PRLR chimeric antibodies (i.e.,antibodies possessing human variable domains and mouse constant domains)were obtained. In addition, several fully human anti-PRLR antibodieswere isolated directly from antigen-positive B cells without fusion tomyeloma cells, as described in US 2007/0280945A1.

Certain biological properties of the exemplary anti-PRLR antibodiesgenerated in accordance with the methods of this Example are describedin detail in the Examples set forth below.

Example 2. Heavy and Light Chain Variable Region Amino Acid and NucleicAcid Sequences

Table 1 sets forth the amino acid sequence identifiers of the heavy andlight chain variable regions and CDRs of selected anti-PRLR antibodiesdescribed herein. The corresponding nucleic acid sequence identifiersare set forth in Table 2.

TABLE 1 Amino Acid Sequence Identifiers Antibody SEQ ID NOs: DesignationHCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H6762P 2 4 6 8 10 12 1416 H1H6765P 18 20 22 24 26 28 30 32 H1H6774P 34 36 38 40 42 44 46 48H1H6781P 50 52 54 56 58 60 62 64 H1H6782P 66 68 70 72 74 76 78 80H1H6783P 82 84 86 88 90 92 94 96 H1H6785P 98 100 102 104 106 108 110 112H1H6790P 114 116 118 120 122 124 126 128 H1H6792P 130 132 134 136 138140 142 144 H1H6793P 146 148 150 152 154 156 158 160 H1H6795P 162 164166 168 170 172 174 176 H1H6797P 178 180 182 184 186 188 190 192H1H6800P 194 196 198 200 202 204 206 208 H1H6801P 210 212 214 216 218220 222 224 H1H6803P 226 228 230 232 234 236 238 240 H1H6804P 242 244246 248 250 252 254 256 H1H6807P 258 260 262 264 266 268 270 272H1M6953N 274 276 278 280 282 284 286 288 H2M6958N2 290 292 294 296 298300 302 304 H2M6959N2 306 308 310 312 314 316 318 320 H2M6960N 322 324326 328 330 332 334 336 H2M6966N 338 340 342 344 346 348 350 352H2M6967N 354 356 358 360 362 364 366 368 H2M6975N 370 372 374 376 378380 382 384 H2M6976N 386 388 390 392 394 396 398 400

TABLE 2 Nucleic Acid Sequence Identifiers Antibody SEQ ID NOs:Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H6762P 1 3 57 9 11 13 15 H1H6765P 17 19 21 23 25 27 29 31 H1H6774P 33 35 37 39 41 4345 47 H1H6781P 49 51 53 55 57 59 61 63 H1H6782P 65 67 69 71 73 75 77 79H1H6783P 81 83 85 87 89 91 93 95 H1H6785P 97 99 101 103 105 107 109 111H1H6790P 113 115 117 119 121 123 125 127 H1H6792P 129 131 133 135 137139 141 143 H1H6793P 145 147 149 151 153 155 157 159 H1H6795P 161 163165 167 169 171 173 175 H1H6797P 177 179 181 183 185 187 189 191H1H6800P 193 195 197 199 201 203 205 207 H1H6801P 209 211 213 215 217219 221 223 H1H6803P 225 227 229 231 233 235 237 239 H1H6804P 241 243245 247 249 251 253 255 H1H6807P 257 259 261 263 265 267 269 271H1M6953N 273 275 277 279 281 283 285 287 H2M6958N2 289 291 293 295 297299 301 303 H2M6959N2 305 307 309 311 313 315 317 319 H2M6960N 321 323325 327 329 331 333 335 H2M6966N 337 339 341 343 345 347 349 351H2M6967N 353 355 357 359 361 363 365 367 H2M6975N 369 371 373 375 377379 381 383 H2M6976N 385 387 389 391 393 395 397 399

Antibodies are typically referred to herein according to the followingnomenclature: Fc prefix (e.g. “H1H,” “H1M,” “H2M,” etc.), followed by anumerical identifier (e.g. “6762,” “6953,” “6958,” etc.), followed by a“P” or “N” suffix, as shown in Tables 1 and 2. Thus, according to thisnomenclature, an antibody may be referred to herein as, e.g.,“H1H6762P,” “H1M6953N,” “H2M6958N,” etc. The H1H, H1M and H2M prefixeson the antibody designations used herein indicate the particular Fcregion isotype of the antibody. For example, an “H1H” antibody has ahuman IgG1 Fc, an “HIM” antibody has a mouse IgG1 Fc, and an “H2M”antibody has a mouse IgG2 Fc, (all variable regions are fully human asdenoted by the first ‘H’ in the antibody designation). As will beappreciated by a person of ordinary skill in the art, an antibody havinga particular Fc isotype can be converted to an antibody with a differentFc isotype (e.g., an antibody with a mouse IgG1 Fc can be converted toan antibody with a human IgG4, etc.), but in any event, the variabledomains (including the CDRs)—which are indicated by the numericalidentifiers shown in Tables 1 and 2—will remain the same, and thebinding properties are expected to be identical or substantially similarregardless of the nature of the Fc domain.

Control Constructs Used in the Following Examples

Control constructs were included in the following experiments forcomparative purposes: Control I: a human anti-PRLR antibody with heavyand light chain variable domains having the amino acid sequences of thecorresponding domains of “he.06.642-2,” as set forth in WO2008/02295A2;and Control II: a human anti-ErbB2 antibody with heavy and light chainvariable domains having the amino acid sequences of the correspondingdomains of “4D5v8” as set forth in: Carter et al., 1992, Proc. Natl.Acad. Sci. USA, 89:4285-4289.

Example 3. Surface Plasmon Resonance Derived Binding Affinities andKinetic Constants of Human Monoclonal Anti-PRLR Antibodies

Binding affinities and kinetic constants of human monoclonal anti-PRLRantibodies were determined by surface plasmon resonance at 25° C. and37° C. Antibodies, expressed as human IgG1 Fc (i.e., “H1H”designations), were captured onto an anti-human Fc sensor surface(mAb-capture format), and soluble monomeric (hPRLR.mmh; SEQ ID NO:401,or macaca fascicularis (mf) PRLR.mmh; SEQ ID NO:403) or dimeric(hPRLR.mFc; SEQ ID NO:402) PRLR protein was injected over the sensorsurface. Measurements were conducted on a T200 Biacore instrument.Kinetic association (k_(a)) and dissociation (k_(d)) rate constants weredetermined by processing and fitting the data to a 1:1 binding modelusing Scrubber 2.0 curve fitting software. Binding dissociationequilibrium constants (K_(D)) and dissociative half-lives (t_(1/2)) werecalculated from the kinetic rate constants as: K_(D) (M)=k_(d)/k_(a);and t_(1/2) (min)=(ln 2/(60*k_(d)). Results are summarized in Tables 3and 4.

TABLE 3 Biacore Binding Affinities of Human Fc mAbs at 25° C. Binding at25° C./Antibody-Capture Format K_(D) T½ Antibody Analyte ka (Ms⁻¹) kd(s⁻¹) (Molar) (min) H1H6953N hPRLR.mmh 7.92E+05 2.12E−04 2.68E−10 54.5hPRLR.mFc 7.11E+05 4.77E−05 6.70E−11 242.2 mfPRLR.mmh 7.27E+05 3.19E−044.38E−10 36.3 H1H6958N2 hPRLR.mmh 2.33E+05 2.72E−04 1.17E−09 42.5hPRLR.mFc 4.22E+05 3.66E−05 8.67E−11 315.8 mfPRLR.mmh 1.95E+05 2.80E−041.44E−09 41.3 H1H6959N2 hPRLR.mmh 1.79E+05 3.60E−02 2.02E−07 0.3hPRLR.mFc 3.59E+05 5.18E−04 1.45E−09 22.3 mfPRLR.mmh 1.29E+05 1.67E−021.29E−07 0.7 H1H6960N hPRLR.mmh 1.08E+05 7.29E−04 6.74E−09 15.8hPRLR.mFc 2.52E+05 3.56E−05 1.41E−10 324.4 mfPRLR.mmh 9.40E+04 5.83E−046.20E−09 19.8 H1H6966N hPRLR.mmh 8.52E+05 2.57E−04 3.02E−10 44.9hPRLR.mFc 9.31E+05 4.08E−05 4.39E−11 282.9 mfPRLR.mmh 7.82E+05 3.25E−044.16E−10 35.6 H1H6967N hPRLR.mmh 2.46E+05 3.32E−04 1.35E−09 34.8hPRLR.mFc 4.07E+05 4.45E−05 1.10E−10 259.3 mfPRLR.mmh 1.90E+05 5.85E−043.08E−09 19.7 H1H6975N hPRLR.mmh 1.50E+05 7.35E−05 4.90E−10 157.1hPRLR.mFc 2.87E+05 1.97E−05 6.84E−11 587.8 mfPRLR.mmh 1.15E+05 1.31E−041.14E−09 88.2 H1H6976N hPRLR.mmh 5.44E+05 8.64E−04 1.59E−09 13.4hPRLR.mFc 1.12E+06 1.01E−04 9.06E−11 114.4 mfPRLR.mmh 4.66E+05 8.14E−041.75E−09 14.2 H1H6762P hPRLR.mmh 6.92E+05 1.79E−04 2.59E−10 64 hPRLR.mFc6.51E+05 6.45E−05 9.92E−11 179 mfPRLR.mmh 4.08E+05 2.08E−04 5.11E−10 55H1H6765P hPRLR.mmh 9.07E+05 1.13E−04 1.24E−10 102 hPRLR.mFc 8.69E+053.41E−05 3.92E−11 339 mfPRLR.mmh 5.27E+05 1.34E−04 2.54E−10 86 H1H6774PhPRLR.mmh 7.15E+05 8.18E−04 1.14E−09 14 hPRLR.mFc 7.98E+05 8.94E−051.12E−10 129 mfPRLR.mmh 4.95E+05 8.75E−04 1.77E−09 13 H1H6781P hPRLR.mmh2.08E+05 1.27E−04 6.10E−10 91 hPRLR.mFc 2.57E+05 6.05E−05 2.36E−10 191mfPRLR.mmh 1.43E+05 1.41E−04 9.86E−10 82 H1H6782P hPRLR.mmh 3.60E+052.47E−04 6.85E−10 47 hPRLR.mFc 3.17E+05 7.13E−05 2.25E−10 162 mfPRLR.mmh2.66E+05 2.79E−04 1.05E−09 41 H1H6783P hPRLR.mmh 2.88E+05 4.13E−041.43E−09 28 hPRLR.mFc 2.64E+05 8.77E−05 3.32E−10 132 mfPRLR.mmh 1.89E+053.41E−04 1.81E−09 34 H1H6785P hPRLR.mmh 3.01E+05 1.71E−04 5.67E−10 68hPRLR.mFc 2.63E+05 6.07E−05 2.31E−10 190 mfPRLR.mmh 2.27E+05 1.67E−047.38E−10 69 H1H6790P hPRLR.mmh 5.65E+05 7.99E−04 1.41E−09 14 hPRLR.mFc5.42E+05 1.04E−04 1.92E−10 111 mfPRLR.mmh 3.89E+05 7.93E−04 2.04E−09 15H1H6792P hPRLR.mmh 3.24E+05 7.94E−04 2.45E−09 15 hPRLR.mFc 3.03E+059.48E−05 3.13E−10 122 mfPRLR.mmh 2.36E+05 9.83E−04 4.17E−09 12 H1H6793PhPRLR.mmh 2.35E+05 3.32E−04 1.41E−09 35 hPRLR.mFc 2.29E+05 7.57E−053.31E−10 153 mfPRLR.mmh 1.77E+05 3.93E−04 2.22E−09 29 H1H6795P hPRLR.mmh1.17E+06 1.77E−03 1.52E−09 7 hPRLR.mFc 1.54E+06 8.41E−05 5.45E−11 137mfPRLR.mmh 8.44E+05 1.97E−03 2.33E−09 6 H1H6797P hPRLR.mmh 1.13E+061.96E−03 1.73E−09 6 hPRLR.mFc 9.82E+05 1.19E−04 1.22E−10 97 mfPRLR.mmh6.70E+05 2.09E−03 3.12E−09 6 H1H6800P hPRLR.mmh 4.21E+05 4.09E−049.72E−10 28 hPRLR.mFc 4.73E+05 8.69E−05 1.84E−10 133 mfPRLR.mmh 3.03E+053.80E−04 1.25E−09 30 H1H6801P hPRLR.mmh 8.46E+05 7.56E−04 8.94E−10 15hPRLR.mFc 6.75E+05 1.09E−04 1.61E−10 106 mfPRLR.mmh 6.57E+05 1.23E−031.88E−09 9 H1H6803P hPRLR.mmh 8.24E+04 1.37E−04 1.67E−09 84 hPRLR.mFc1.04E+05 6.29E−05 6.07E−10 184 mfPRLR.mmh 6.21E+04 2.09E−04 3.37E−09 55H1H6804P hPRLR.mmh 4.53E+05 6.34E−04 1.40E−09 18 hPRLR.mFc 4.51E+058.69E−05 1.93E−10 133 mfPRLR.mmh 3.31E+05 6.57E−04 1.99E−09 18 H1H6807PhPRLR.mmh 7.61E+05 1.44E−04 1.89E−10 80 hPRLR.mFc 6.80E+05 5.46E−058.03E−11 212 mfPRLR.mmh 4.37E+05 1.51E−04 3.46E−10 76 Control IhPRLR.mmh 5.11E+05 7.44E−04 1.46E−09 15.5 hPRLR.mFc 4.72E+05 7.53E−051.59E−10 153.5 mfPRLR.mmh 2.38E+05 6.14E−03 2.59E−08 1.9 NB = No bindingobserved under conditions used

TABLE 4 Biacore Binding Affinities of Human Fc mAbs at 37° C. Binding at37° C./Antibody-Capture Format K_(D) T 1/2 Antibody Analyte ka (Ms⁻¹) kd(s⁻¹) (Molar) (min) H1H6953N hPRLR.mmh 1.10E+06 1.22E−03 1.11E−09 9.4hPRLR.mFc 1.47E+06 1.70E−04 1.16E−10 68.0 mfPRLR.mmh 9.47E+05 2.56E−032.71E−09 4.5 H1H6958N2 hPRLR.mmh 4.13E+05 1.31E−03 3.16E−09 8.8hPRLR.mFc 8.29E+05 1.39E−04 1.67E−10 83.3 mfPRLR.mmh 3.28E+05 1.34E−034.08E−09 8.6 H1H6959N2 hPRLR.mmh 4.06E+04 2.77E−02 6.81E−07 0.4hPRLR.mFc 5.09E+05 2.30E−03 4.51E−09 5.0 mfPRLR.mmh 4.46E+04 1.65E−023.70E−07 0.7 H1H6960N hPRLR.mmh 1.22E+05 1.98E−03 1.62E−08 5.8 hPRLR.mFc2.94E+05 1.47E−04 5.00E−10 78.7 mfPRLR.mmh 8.64E+04 1.28E−03 1.49E−089.0 H1H6966N hPRLR.mmh 1.58E+06 9.60E−04 6.07E−10 12.0 hPRLR.mFc1.88E+06 1.27E−04 6.72E−11 91.3 mfPRLR.mmh 1.22E+06 1.20E−03 9.82E−109.6 H1H6967N hPRLR.mmh 4.24E+05 9.33E−04 2.20E−09 12.4 hPRLR.mFc9.07E+05 9.73E−05 1.07E−10 118.8 mfPRLR.mmh 3.56E+05 1.62E−03 4.54E−097.1 H1H6975N hPRLR.mmh 2.11E+05 2.73E−04 1.29E−09 42.3 hPRLR.mFc3.86E+05 7.09E−05 1.84E−10 163.0 mfPRLR.mmh 1.40E+05 3.17E−04 2.27E−0936.4 H1H6976N hPRLR.mmh 7.77E+05 3.14E−03 4.04E−09 3.7 hPRLR.mFc1.37E+06 1.40E−04 1.02E−10 82.6 mfPRLR.mmh 6.03E+05 3.16E−03 5.24E−093.7 H1H6762P hPRLR.mmh 9.48E+05 4.55E−04 4.80E−10 25 hPRLR.mFc 8.01E+051.03E−04 1.28E−10 112 mfPRLR.mmh 6.79E+05 5.58E−04 8.23E−10 21 H1H6765PhPRLR.mmh 1.25E+06 3.66E−04 2.92E−10 32 hPRLR.mFc 8.01E+05 1.03E−041.28E−10 112 mfPRLR.mmh 1.06E+06 5.37E−05 5.04E−11 215 H1H6774PhPRLR.mmh 1.07E+06 3.17E−03 2.95E−09 4 hPRLR.mFc 1.41E+06 1.94E−041.38E−10 60 mfPRLR.mmh 7.23E+05 3.61E−03 5.00E−09 3 H1H6781P hPRLR.mmh3.39E+05 3.09E−04 9.10E−10 37 hPRLR.mFc 4.36E+05 9.84E−05 2.26E−10 117mfPRLR.mmh 2.47E+05 2.67E−04 1.08E−09 43 H1H6782P hPRLR.mmh 5.57E+057.16E−04 1.28E−09 16 hPRLR.mFc 5.98E+05 1.30E−04 2.17E−10 89 mfPRLR.mmh3.85E+05 7.67E−04 1.99E−09 15 H1H6783P hPRLR.mmh 4.11E+05 1.61E−033.91E−09 7 hPRLR.mFc 4.91E+05 1.38E−04 2.82E−10 83 mfPRLR.mmh 2.73E+051.30E−03 4.74E−09 9 H1H6785P hPRLR.mmh 4.26E+05 4.84E−04 1.14E−09 24hPRLR.mFc 4.56E+05 1.17E−04 2.56E−10 99 mfPRLR.mmh 2.97E+05 4.50E−041.52E−09 26 H1H6790P hPRLR.mmh 9.40E+05 3.29E−03 3.50E−09 4 hPRLR.mFc6.46E+05 1.98E−04 3.06E−10 58 mfPRLR.mmh 6.15E+05 3.21E−03 5.22E−09 4H1H6792P hPRLR. mmh 4.35E+05 2.29E−03 5.27E−09 5 hPRLR.mFc 4.99E+051.76E−04 3.52E−10 66 mfPRLR.mmh 3.05E+05 2.86E−03 9.37E−09 4 H1H6793PhPRLR.mmh 3.39E+05 1.02E−03 3.02E−09 11 hPRLR.mFc 4.10E+05 1.42E−043.47E−10 81 mfPRLR.mmh 2.33E+05 1.07E−03 4.59E−09 11 H1H6795P hPRLR.mmh1.36E+06 5.20E−03 3.81E−09 2 hPRLR.mFc 1.94E+06 7.77E−05 4.02E−11 149mfPRLR.mmh 9.73E+05 5.99E−03 6.16E−09 2 H1H6797P hPRLR.mmh 1.29E+068.22E−03 6.37E−09 1 hPRLR.mFc 1.80E+06 1.25E−04 6.91E−11 93 mfPRLR.mmh9.14E+05 9.06E−03 9.90E−09 1 H1H6800P hPRLR.mmh 8.08E+05 1.19E−031.47E−09 10 hPRLR.mFc 6.44E+05 1.47E−04 2.29E−10 79 mfPRLR.mmh 4.39E+051.09E−03 2.48E−09 11 H1H6801P hPRLR.mmh 9.51E+05 4.41E−03 4.63E−09 3hPRLR.mFc 7.93E+05 2.21E−04 2.79E−10 52 mfPRLR.mmh 7.11E+05 7.71E−031.08E−08 1 H1H6803P hPRLR.mmh 1.29E+05 3.64E−04 2.83E−09 32 hPRLR.mFc1.34E+05 6.20E−05 4.61E−10 186 mfPRLR.mmh 8.73E+04 6.36E−04 7.28E−09 18H1H6804P hPRLR.mmh 6.07E+05 3.85E−03 6.34E−09 3 hPRLR.mFc 5.54E+052.05E−04 3.69E−10 56 mfPRLR.mmh 4.55E+05 4.26E−03 9.35E−09 3 H1H6807PhPRLR.mmh 1.08E+06 3.36E−04 3.10E−10 34 hPRLR.mFc 1.22E+06 1.10E−049.00E−11 105 mfPRLR.mmh 8.02E+05 3.59E−04 4.48E−10 32 Control IhPRLR.mmh 5.99E+05 2.42E−03 4.04E−09 4.8 hPRLR.mFc 8.47E+05 2.14E−042.53E−10 54.0 mfPRLR.mmh 1.53E+05 1.54E−02 1.01E−07 0.8 NB = No bindingobserved under conditions used

As shown in Tables 3 and 4, several antibodies described hereindisplayed sub-nanomolar affinities to human and monkey PRLR protein andexhibited higher affinity than the comparator anti-PRLR antibody(Control I). For example, at 37° C., many of the anti-PRLR antibodiesbound to monomeric human PRLR with K_(D) values less than 4 nM and T½times greater than 5 minutes; and to dimeric human PRLR with K_(D)values less than 250 pM and T^(1/2) times greater than 60 minutes. Thesebinding characteristics are substantially better than what was observedwith the Control I antibody under the same experimental conditions.

Example 4A. Anti-PRLR Antibodies Bind to Endogenous and OverexpressedPRLR Cell Lines

The ability of anti-PRLR antibodies to selectively bind PRLR expressingcell lines was next determined. Human, monkey macaca fascicularis, andmouse PRLR constructs with an HA tag were stably introduced into HEK293cells via Lipofectamine 2000-mediated transfection methodology.Transfectants (HEK293/hPRLR, HEK293/mfPRLR and HEK293/mPRLR) wereselected for at least 2 weeks in complete growth media plus G418.

Cell surface expression of PRLR on 293/hPRLR cells was assessed via FACSanalysis. Briefly, 1×10⁵ cells were incubated with 10 μg/ml of Controlantibody I, or an isotype control for 30 min on ice in antibody dilutionbuffer. Following two washes with antibody dilution buffer, cells wereincubated with 10 μg/ml of PE conjugated anti-human secondary antibodiesfor 30 min on ice. Following two additional washes, samples were run ona Hypercyt® cytometer and analyzed in ForeCyt™ (IntelliCyt, Albuquerque,N. Mex.). The mean fluorescence intensities (MFI) were expressed as foldchange above isotype control levels (background). FACS binding confirmedthat Control I selectively bound to 293/hPRLR expressing cells with MFIsthat were 30 fold above background (isotype ctrl) levels and less than 2fold binding on parental cells.

Cell surface copy number of PRLR was also quantitatively determined onT47D, MCF7 and MCF7/hPRLR-overexpressing cell lines. Briefly, 1×10⁵cells were incubated with 100 nM of the anti-PRLR antibodyH1H6953N-Alexa647 for 30 min on ice in antibody dilution buffer.Following two washes with antibody dilution buffer, samples were run ona Hypercyt® cytometer (IntelliCyt, Albuquerque, N. Mex.) and the meanfluorescence intensities (MFI) were determined in ForeCyt™ (IntelliCyt,Albuquerque, N. Mex.). The MFI for each cell line was then converted toAlexa647 molecules of equivalent soluble fluorescence (MESF) via theQuantum Alexa Fluor 647 MESF kit according to manufacturer instructions(Bangs Laboratories, Inc, Fishers, Ind.). The average number offluorophores per H1H6953N-A647 protein (F/P ratio) was determined viathe Simply Cellular anti-Human IgG kit according to manufactureinstructions (Bangs Laboratories, Inc, Fishers, Ind.). The MESF valueswere divided by the F/P ratio to determine the PRLR cell surface copynumber or H1H6953N antigen binding capacity on each cell line. Usingthis method, it was determined that the approximate cell surface copynumber of PRLR on the various cell lines was as follows: T47D=27,000;MCF7=3,000; and MCF7/hPRLR=190,000.

Next, the anti-PRLR antibodies were tested via FACS for selectivebinding to the engineered overexpressing PRLR HEK293 cell lines, as wellas to non-expressing HEK293 cells and a native PRLR expressing cell line(T47D). Results are shown in Table 5.

TABLE 5 FACS Cell Surface Binding of Anti-PRLR Antibodies FACS CellBinding (Fold Above Background) HEK293/ HEK293/ HEK293/ Antibody HEK293hPRLR mfPRLR mPRLR T47D unstained 1 1 1 1 1 Secondary 1 1 1 1 1 onlyControl I 1 31 13 7 30 *H1H6762P 1 36 26 2 38 *H1H6765P 2 38 27 1 40H1H6774P 1 1 29 1 39 *H1H6781P 1 37 26 1 40 H1H6782P 3 41 32 4 43*H1H6783P 1 37 27 1 38 *H1H6785P 1 37 26 2 40 *H1H6790P 1 37 24 1 34*H1H6792P 1 37 28 3 37 H1H6793P 2 2 32 2 43 H1H6795P 1 1 22 1 34H1H6797P 1 1 25 2 38 *H1H6800P 1 35 29 1 41 H1H6801P 7 47 88 56 45*H1H6803P 1 39 28 1 39 H1H6804P 1 1 29 1 34 *H1H6807P 2 32 27 2 34*H1H6953N 2 37 29 2 40 *H1H6958N2 1 35 30 1 37 H1H6959N2 1 6 11 2 9*H1H6960N 1 29 23 1 33 *H1H6966N 1 29 18 1 31 *H1H6967N 1 38 28 2 41*H1H6975N 1 38 29 1 46 H1H6976N 1 8 29 1 37 Isotype Ctrl I 1 1 1 1 NAIsotype Ctrl II 1 1 NA NA 1 *Denotes antibodies with specific binding onHEK293/hPRLR, HEK293/mfPRLR and T47D and less than 2-fold binding onHEK293 parental cells.

As shown in Table 5, a majority of the anti-human PRLR antibodiesspecifically bound to HEK293/PRLR cells at >25-fold above backgroundlevels with negligible binding to parental cells. Antibodies that boundto human PRLR were similarly shown to bind to monkey (macacafascicularis) PRLR on HEK293/mfPRLR cells. Antibodies that wereidentified to be strong binders to HEK293/hPRLR cells were similarlyshown to be robust binders to native PRLR expressing T47D cells.Cross-reactivity to rodent PRLR was not observed.

In summary, antibodies displayed strong binding to human and monkey PRLRon engineered cell lines as well as endogenously expressed PRLR.

Example 4B. Anti-PRLR Antibodies are Internalized by PRLR-ExpressingCells In Vitro

In this Example, the internalization of anti-PRLR antibodies byPRLR-expressing cells (T47D) was assessed. Briefly, 20,000 T47D cellswere seeded in collagen coated 96 well plates. The next day, cells wereincubated with anti-human PRLR antibodies (10 μg/ml) for 30 min on icefollowed by two PBS washes. Cells were then incubated with alexa488conjugated anti-hFc Fab secondary antibodies for 30 minutes on icefollowed by two additional PBS washes. Antibodies were allowed tointernalize for 1 h at 37° C. in internalization buffer (PBS+2% FBS) orremained at 4° C. Cells were fixed in 4% formaldehyde, nuclei werestained with DRAQ5 (Cell signaling), and images were acquired on theImageXpress micro XL (Molecular Devices). Whole cell alexa488 intensityat 37° C. (Binding) and the alexa488 intensity in the intracellularvesicles at 37° C. (Internalization) were determined via Columbus imageanalysis software (PerkinElmer). The intensities are expressed as apercentage of the strongest internalizing antibody, H1H6975N, and aresummarized in Table 6.

TABLE 6 Cell Line: T47D [mAb] 1 μg/mL (0.67 nM) % % TotalInternalization Binding relative to relative to Antibody Control IControl I anti-PRLR 100.0 100.0 control I H1H6975N 214.2 225.9 H1H6800P198.3 186.8 H1H6803P 190.5 205.5 H1H6762P 186.3 176.7 H1H6765P 186.3191.3 H1H6793P 179.9 177.9 H1H6782P 179.3 209.9 H1H6976N 169.5 180.4H1H6785P 169.1 168.0 H1H6958N2 169.0 161.5 H1H6967N 168.6 158.6 H1H6781P165.1 166.2 H1H6774P 162.3 173.7 H1H6783P 160.7 165.8 H1H6792P 155.9110.6 H1H6953N 153.9 164.2 H1H6795P 150.7 123.4 H1H6801P 148.9 155.7H1H6807P 147.0 152.7 H1H6790P 146.9 122.8 H1H6797P 145.2 151.2 H1H6804P138.9 149.6 H1H6966N 137.2 111.3 H1H6960N 120.0 89.7 H1H6959N2 15.3 2.0

With the exception of H1H6959N2, all tested antibodies bound T47D andnearly 100% of all bound mAbs internalized within 1 h. The totalinternalized antibody intensity for most antibodies was greater than theanti-human PRLR control antibody (Control I).

Example 5. Anti-PRLR Antibodies Inhibit PRL-Mediated Receptor Activationin Cells Expressing Human PRLR

The ability of anti-PRLR antibodies to block prolactin (PRL)-mediatedreceptor activation was examined in a luciferase-based reporter assay.The endocrine hormone PRL binds to the extracellular domain of itscognate receptor PRLR, triggering rapid homodimerization and activatingseveral downstream signaling cascades.

In this example, an engineered cell line was used to determine theability of anti-PRLR antibodies to block ligand activation of the PRLRreceptor. Briefly, HEK293/hPRLR/STAT5-Luc cell lines with stableincorporation of a human PRLR expression cassette and theSTAT5-dependent luciferase reporter were generated via sequential roundsof Lipofectamine® 2000-mediated transfection (LifeTechnologies,Carlsbad, Calif.). Cells were selected for at least two weeks in thepresence of 500 μg/mL G418 (hPRLR) and 100 μg/mL hygromycin B(STAT5-Luc). The STAT5-Luc assay utilized 2×10⁵ HEK293/hPRLR/STAT5-Luccells seeded in complete growth medium on PDL-coated 96 well platesgrown overnight at 37° C., 5% CO₂. To generate antibody inhibitioncurves, cells were incubated (6 hr at 37° C.) with serially dilutedanti-human PRLR antibodies (100 nM to 24 pM) in the presence of 5 nMconstant PRL before recording signal. PRL dose response curves weregenerated by the addition of serially diluted PRL (100 nM to 24 pM) tocells and recording signal after a 6 hr (37° C.) incubation in theabsence of antibodies. The ability of the antibodies to activate PRLR inthe absence of ligand was also assessed.

Luciferase activity was measured with ONE-Glo™ reagent (Promega,Madison, Wis.). Relative light units (RLUs) were measured on a Victorluminometer (Perkin Elmer, Shelton, Conn.). EC₅₀/IC₅₀ values weredetermined from a four-parameter logistic equation over an 8-pointresponse curve using GraphPad Prism. Percent blocking and percentactivation are reported for the highest antibody dose. Results are shownin Table 7.

TABLE 7 IC₅₀ and Percent Blocking of PRL-Mediated Signaling by Anti-PRLRAntibodies Percent Blocking IC₅₀ of Blocking at 100 nM Antibody 5 nM PRL[M] Antibody H1H6762P 2.19E−11 100 H1H6765P 3.30E−11 100 H1H6774P NB 0H1H6781P 2.70E−11 100 H1H6782P NB 0 H1H6783P ND 59 H1H6785P 3.45E−10 100H1H6790P 2.06E−10 100 H1H6792P 5.70E−10 100 H1H6793P ND 65 H1H6795P NB 0H1H6797P NB 0 H1H6800P 3.55E−10 100 H1H6801P 1.40E−10 100 H1H6803P6.27E−10 100 H1H6804P 7.89E−09 100 H1H6807P 6.00E−10 100 H1H6953N1.05E−10 100 H1H6958N2 1.98E−10 100 H1H6959N2 ND 52 H1H6960N 1.58E−09100 H1H6966N ND 54 H1H6967N 7.68E−10 100 H1H6975N 2.41E−10 100 H1H6976NND 23 Control I 1.33E−09 100 NB: Not blocking; ND: Not determined due toincomplete blocking

As summarized in Table 7, a majority of the antibodies inhibitedactivation of the STAT5 reporter, with IC₅₀ values ranging from 22 pM to8 nM. All inhibitory antibodies blocked activation to baseline levels(100 percent blocking). Additionally, the antibodies tested in thisassay did not activate STAT5 in the absence of PRL ligand.

In summary, the data of this Example show that a majority of theanti-PRLR antibodies block PRL-mediated receptor activation.Additionally, a majority of the antibodies inhibit receptor activationmore potently than the anti-PRLR Control I antibody. For example,several anti-PRLR antibodies blocked prolactin-mediated signaling withIC₅₀ values of less than about 1.3 nM. On the other hand, certainanti-PRLR antibodies, despite being able to bind PRLR, did not exhibitprolactin blocking activity. Such non-blocking anti-PRLR antibodies mayfind uses in various therapeutic contexts where PRLR targeting isdesired without interfering with normal prolactin-mediated signaling.

Example 6. Preparation and Characterization of Anti-PRLR Antibody DrugConjugates

Selected anti-PRLR antibodies were conjugated to the maytansinoid DM1through an SMCC linker using methods similar to those set forth in U.S.Pat. No. 5,208,020 and US Patent Application Publication No.2010/0129314, the disclosures of which are incorporated by referenceherein in their entireties. The conjugates were purified by sizeexclusion chromatography and sterile filtered. All starting materialsand solvents were purchased commercially and used without purification,unless otherwise noted.

Protein and linker/payload concentrations were determined by UV spectralanalysis and MALDI-TOF mass spectrometry. Size-exclusion HPLCestablished that all conjugates used were >95% monomeric, and RP-HPLCestablished that there was <0.5% unconjugated linker payload. Yields arereported in Table 8 and 9 based on protein. All conjugated antibodieswere analyzed by UV for linker payload loading values according toHamblett et al, 2004, Clinical Cancer Research 10(20):7063-7070, and bymass difference, native versus conjugated.

TABLE 8 Protein Concentrations for Anti-PRLR Unconjugated Antibodiesε252 nm ε280 nm (cm⁻¹ M⁻¹) (cm⁻¹ M⁻¹) Compound SMCC-DM1 26790 5700Antibody (unconjugated) H1H6958N2 74462 195440 H1H6959N2 77485 209420H1H6960N 84926 214460 H1H6953N 80673 220420 H1H6975P 81120 199804Isotype Control 84723 218360

TABLE 9 Antibody Linker/Payload Concentrations for Anti-PRLR-SMCC-DM1Conjugates Antibody Payload:Antibody Payload:Antibody Conjugate MolarRatio (UV) Molar Ratio (MS) Yield % H1H6958N2-DM1 4.0 3.4 64H1H6959N2-DM1 3.8 3.3 64 H1H6960N-DM1 3.6 3.0 64 H1H6953N-DM1 3.2 2.7 52H1H6803P-DM1 ND 3.1 55 H1H6762P-DM1 ND 2.9 70 H1H6765P-DM1 ND 2.3 55H1H6782P-DM1 ND 2.8 65 H1H6793P-DM1 ND 3.8 55 H1H6975P-DM1 3.0 3.4 60H1H6800P-DM1 3.0 3.2 50 Isotype Control-DM1 3.3 3.3 80 ND: notdetermined

This Example illustrates the conjugation of anti-PRLR antibodies withDM1 through an SMCC linker. The payload: antibody molar ratio wascalculated to be from about 2.3 to about 3.8 for the conjugatedantibodies of this Example. Percent yields for the antibodies rangedfrom around 50% to 70%.

Example 7. Anti-PRLR Antibody-Drug Conjugates Effectively Kill Cellswith Low-to-Moderate PRLR Expression Levels as Well as Cells with HighPRLR Expression Levels

To determine the relative cell-killing potency of anti-PRLR ADCscompared to a similar anti-ErbB2 ADC, cell-killing assays were run onmultiple cells lines expressing either PRLR, ErbB2 or a combination ofboth receptors.

PRLR-overexpressing cells, including HEK293, PC3, MCF7 and NCI-N87, weregenerated to assess the ability of anti-PRLR conjugated antibodies toreduce cell viability. For comparative purposes, PC3 and T47D cells withoverexpressed ErbB2 were also generated, as well as an MCF7 cell lineover-expressing both hPRLR and hErbB2. Briefly, Lipofectamine®2000-mediated transfection methodology was utilized to generate HEK293cells expressing human PRLR (HEK293/hPRLR) or human ErbB2(HEK293/hErbB2). Lipofectamine LTX with Plus Reagent was used togenerate PC3 cells expressing human PRLR (PC3/hPRLR) or human ErbB2(PC3/hErbB2). Lentiviral-mediated transduction was utilized to generateMCF7 cells expressing human PRLR (MCF7/hPRLR), NCI-N87 cells expressinghuman PRLR (NCI-N87/hPRLR), T47D cells over-expressing human ErbB2(T47D/hErbB2), and MCF7 cells expressing both human PRLR and human ErbB2(MCF7/hPRLR/hErbB2). All lines were selected for at least two weeks incomplete growth media plus appropriate selection reagents. Stablyexpressing populations were enriched for PRLR expression via FACS usingthe anti PRLR antibody Control I.

Cell surface expression of PRLR and ErbB2 was analyzed via FACS usingeither the Control I anti-PRLR antibody or Control II anti-HER2antibody, respectively. Additionally, endogenous PRLR cell surfaceexpression on the T47D#11 cell line, a variant of the T47D line selectedfor more aggressive in vivo tumor growth, was also determined.Approximately 1×10⁶ cells were incubated with 10 μg/ml of anti-PRLRControl Antibody (Control I), an anti-ErbB2 control antibody (ControlII), or an isotype control for 30 min on ice in antibody dilutionbuffer. Following two washes with antibody dilution buffer, cells wereincubated with 10 μg/ml of PE conjugated anti-human secondary antibodiesfor 30 min on ice. Following two additional washes, samples were run onthe Accuri C6 (BD) cytometer and analyzed with FlowJo software (TreeStar, Inc., Ashland, Oreg.). Relative expression level results are shownin Table 10.

TABLE 10 Human PRLR Cell Surface Expression (Engineered & EndogenousLines) FACS Binding (MFI FOLD ABOVE ISOTYPE CONTROL) Sec- Anti- ondaryIsotype PRLR Anti-ErbB2 Cell Line Unstained alone Ctrl (Control I)(Control II) 293 1X 1X 1X 1X 28X 293/hErbB2 1X 1X 1X 1X 215X 293/hPRLR1X 1X 1X 18X 18X PC3 1X 1X 1X 1X 41X PC3/hErbB2 1X 1X 1X 1X 238XPC3/hPRLR 1X 1X 1X 13X 31X T47D 1X 1X 1X 12X 87X T47D#11 1X 1X 1X 10X NDT47D/hErbB2 1X 1X 1X 12X 437X SK-BR-3 1X 1X 1X 1X 600X MCF7 1X 1X 1X 3X42X MCF7/hPRLR 1X 1X 1X 55X 36X MCF7/hPRLR/ 1X 1X 1X 55X 349X hErbB2NCI-N87 1X 1X 1X 1X 1,400X NCI-N87/ 1X 1X 1X 6X 1,400X hPRLR

In general, exogenous PRLR surface expression ranged from 6-fold to55-fold over background, with most engineered cells exhibiting 12-foldto 18-fold PRLR expression over background. Endogenous PRLR expressionwas 3-fold over background in MCF7 cells but was not detected inparental HEK293, PC3 and NCI-N87 lines. Endogenous PRLR expression was12-fold over background in the T47D cell line and 10-fold overbackground in the T47D#11 variant cell line. ErbB2 expression wasdetected in all PRLR-expressing cell lines, and ranged from 18-fold to1400-fold above background.

Next, the ability of anti-PRLR-DM1 antibody-drug conjugates (i.e.,anti-PRLR antibodies conjugated to DM1 via a non-cleavable linker[SMCC]) to reduce cell viability was determined using in vitro cellbased assays. Cells were seeded in PDL-coated 96 well plates at 1500 to10000 cells per well in complete growth media and allowed to growovernight. For cell viability curves, ADCs or free DM1 (as the methyldisulfide derivative DM1-SMe) were added to the cells at finalconcentrations ranging from 500 nM to 5 pM and incubated for 3 days. The293, PC3 and T47D cells were incubated with CCK8 (Dojindo, Rockville,Md.) for the final 1-3 hours and the absorbance at 450 nm (OD₄₅₀) wasdetermined on a Flexstation3 (Molecular Devices, Sunnyvale, Calif.).MCF7 cells were treated with Hoechst 33342 nuclear stain while beingfixed with 4% formaldehyde. Images were acquired on the ImageXpressmicro XL (Molecular Devices, Sunnyvale, Calif.) and nuclear counts weredetermined via Columbus image analysis software (Perkin Elmer, Shelton,Conn.). Background OD₄₅₀ values (PC3, 293, and T47D) or nuclear counts(MCF7) from digitonin (40 nM) treated cells was subtracted from allwells and viability was expressed as a percentage of the untreatedcontrols. IC₅₀ values were determined from a four-parameter logisticequation over a 10-point response curve (GraphPad Prism). IC₅₀ valuesand percent cell killing are shown in Tables 11 and 12.

TABLE 11 Cell Kill Potency of Anti-PRLR-DM1 Antibody-Drug ConjugatesAntibody-Drug 293 293/PRLR PC3 PC3/PRLR Conjugate IC₅₀ % Kill IC₅₀ %Kill IC₅₀ % Kill IC₅₀ % Kill DM1 (free drug) 0.27 98 0.36 97 0.47 910.59 80 H1H6953N-DM1 100 88 0.28 95 110 78 0.86 67 H1H6958N2-DM1 75 980.10 95 150 83 0.43 83 H1H6959N2-DM1 75 100 4.82 95 150 79 11.6 82H1H6960N-DM1 75 100 0.38 95 150 80 1.13 82 H1H6975N-DM1 100 87 ND ND 20071 2.70 68 H1H6762P-DM1 100 89 ND ND 250 78 0.98 66 H1H6765P-DM1 100 86ND ND 200 70 0.57 70 H1H6782P-DM1 100 86 ND ND 300 67 0.92 64H1H6793P-DM1 100 84 ND ND 200 72 3.65 65 H1H6800P-DM1 150 85 ND ND 15072 1.37 68 H1H6803P-DM1 300 7 ND ND 150 72 2.32 70 Control I-DM1 100 920.28 95 >100 79 7.28 80 Isotype ctrl-DM1 100 60 100 93 125 78 110 63IC₅₀ values are in nM; ND: not determined

TABLE 12 Cell Kill Potency of Anti-PRLR-DM1 Antibody-Drug Conjugates(continued) T47D MCF7 MCF7/PRLR Antibody-Drug % % % Conjugate IC₅₀ KillIC₅₀ Kill IC₅₀ Kill DM1 (free drug) 0.45 86 1.17 83 1.47 88 H1H6953N-DM11.64 79 100 55 0.29 76 H1H6958N2-DM1 1.34 71 100 77 0.19 77H1H6959N2-DM1 48.90 71 100 82 0.47 80 H1H6960N-DM1 5.10 71 100 77 0.2978 H1H6975N-DM1 6.90 80 150 73 0.49 87 H1H6762P-DM1 12.60 80 110 64 0.4683 H1H6765P-DM1 1.63 78 150 68 0.18 84 H1H6782P-DM1 4.19 74 120 57 0.6585 H1H6793P-DM1 11.10 53 125 67 0.54 87 H1H6800P-DM1 3.20 79 150 65 0.4085 H1H6803P-DM1 8.61 77 150 60 0.44 84 Control I-DM1 24.50 65 100 460.59 75 Isotype ctrl-DM1 150 61 120 66 100 84 IC₅₀ values are in nM

As summarized in Tables 11 and 12, several anti-PRLR-DM1 antibody-drugconjugates potently reduced cell viability in multiple cell backgrounds,with IC₅₀ values as low as 100 pM. An exemplary anti-PRLR conjugatedantibody, H1H6958N2-DM1, reduced the cell viability of HEK293, PC3 andMCF7-PRLR expressing cells with sub-nM IC₅₀s ranging from 100 pM to 460pM, and killed endogenously expressing T47D cells with an IC₅₀ of 1.3nM. The similarly conjugated anti-PRLR Control I antibody (ControlI-DM1) was several fold less potent than H1H6958N2-DM1 across all celllines. Non-binding isotype controls and unconjugated antibodies had noimpact on cell viability.

Additionally, the impact of the PRLR ligand, PRL, on PRLR-SMCC-DM1 cellkill in T47D cells was assessed. T47D cells were incubatedsimultaneously with PRL (15 nM) and either a non-blocking anti PRLRantibody (H1H6782P) or a receptor blocking antibody (H1H6958N2). Resultsare summarized in Table 13.

TABLE 13 Cell Kill Potency of Anti-PRLR-DM1 Antibody-Drug Conjugates inthe Presence of PRLR Ligand (PRL) HEK293 T47D IC₅₀ % IC₅₀ % Treatment(nM) Kill (nM) Kill Me-SS-May 0.6 94 0.30 100 Free DM1 IsotypeControl-SMCC-DM1 100 78 300 94 Isotype Control-SMCC-DM1 + 170 82 97 8915 nM PRL H1H6958N2-SMCC-DM1 90 88 1.0 100 H1H6958N2-SMCC-DM1 + 110 863.0 100 15 nM PRL H1H6782P-SMCC-DM1 90 83 1.0 98 H1H6782P-SMCC-DM1 + 7078 2.0 97 15 nM PRL

As shown in Table 13, the presence of PRL had only a modest impact onPRLR ADC-mediated cell kill with an observed 2-3 fold reduction in thecell kill potency of the tested mAbs.

The potency of anti-PRLR conjugated antibodies compared with a similarlyconjugated antibody to the co-expressed ErbB2 cell surface target wasalso assessed. Both PRLR and ErbB2 are expressed in a majority of breastcancers, and anti-ErbB2 antibodies conjugated to DM1 have shown clinicalefficacy in targeting ErbB2 (+) breast cancer (Hurvitz et al; 2013). AnErbB2 Control Antibody (Control II) conjugated to DM1 (Control II-DM1)was tested in in vitro viability assays in the cell lines generatedabove. Cell kill potency of conjugated anti-PRLR antibodies compared tothe anti-ErbB2 conjugated antibody is summarized in Tables 14-17.

TABLE 14 Cell Kill Potency of Anti-PRLR-DM1 and Anti-ErbB2-DM1Antibody-Drug Conjugates Antibody-Drug 293 293/ErbB2 293/PRLR ConjugateIC₅₀ % Kill IC₅₀ % Kill IC₅₀ % Kill DM1 0.5 95 0.26 100 0.82 95 (freedrug) H1H6953N-DM1 100 92 120 98 0.43 93 (Anti PRLR- DM1) Control II-DM1100 89 2.0 98 100 88 (Anti ErbB2- DM1) Isotype Control- 150 86 110 98150 86 DM1 PRLR expression  1X  1X 18X ErbB2 expression 28X 215X 18XIC₅₀ values are in nM

TABLE 15 Cell Kill Potency of Anti-PRLR-DM1 and Anti-ErbB2-DM1Antibody-Drug Conjugates (continued) Antibody-Drug PC3 PC3/ErbB2PC3/PRLR Conjugate IC₅₀ % Kill IC₅₀ % Kill IC₅₀ % Kill DM1 0.44 88 0.4285 0.24 82 (free drug) H1H6953N-DM1 100 80 80 73 0.68 76 (Anti PRLR-DM1) Control II-DM1 90 80 1.1 80 100 62 (Anti ErbB2- DM1) IsotypeControl- 85 82 90 71 100 62 DM1 PRLR expression  1X  1X 13X ErbB2expression 41X 238X 31X IC₅₀ values are in nM

TABLE 16 Cell Kill Potency of Anti-PRLR-DM1 and Anti-ErbB2-DM1Antibody-Drug Conjugates (continued) Antibody-Drug T47D T47D/ErbB2SK-BR-3 MCF7 Conjugate IC₅₀ % Kill IC₅₀ % Kill IC₅₀ % Kill IC₅₀ % KillDM1 0.21 80 0.18 71 0.39 74 0.54 78 (free drug) H1H6953N-DM1 1.3 78 2.480 100 77 82 74 (Anti PRLR-DM1) Control II-DM1 100 59 1.18 81 0.48 81 8068 (Anti ErbB2-DM1) Isotype Control- 100 62 120 75 110 76 100 65 DM1PRLR expression 12X  12X  1X  3X ErbB2 expression 87X 437X 600X 42X IC₅₀values are in nM

TABLE 17 Cell Kill Potency of Anti-PRLR-DM1 and Anti-ErbB2-DM1Antibody-Drug Conjugates (continued) MCF/PRLR + NCI- Antibody-DrugMCF7/PRLR ErbB2 NCI-N87 N87/PRLR Conjugate IC₅₀ % Kill IC₅₀ % Kill IC₅₀% Kill IC₅₀ % Kill DM1 1.85 79 0.82 77 0.84 95 1.45 98 (free drug)H1H6953N-DM1 0.33 76 0.33 77 90 83 2.9 94 (Anti PRLR-DM1) Control II-DM1100 56 0.63 76 0.22 95 0.66 94 (Anti ErbB2-DM1) Isotype Control- 150 61100 70 90 85 85 88 DM1 PRLR expression 55X  55X   1X   6X ErbB2expression 36X 349X 1400X 1400X IC₅₀ values are in nM

Anti-PRLR-DM1 antibodies effectively killed cells even with relativelylow levels of PRLR expression. For example, H1H6953N-DM1 (anti-PRLR-DM1)inhibited the growth of T47D cells (expressing PRLR at only 12× abovebackground) with an IC₅₀ of 1.3 nM and showed 78% killing. This sameantibody also inhibited the growth of 293/hPRLR cells (expressing PRLRat 18× above background) with and IC₅₀ of 0.43 nM and showed 93%killing. Equivalent killing with the anti-ErbB2-DM1 antibody (“controlII”) was observed only in cells that express the target antigen atlevels greater than about 200× to about 400× above background (see e.g.,PC3/hErbB2, expressing ErbB2 at 238× above background and T47D/hErbB2,expressing ErbB2 at 437× above background). Therefore, these datasuggest that anti-PRLR antibody-drug conjugates can effectively targetand kill tumor cells with relatively low levels of PRLR expression,while anti-ErbB2 antibody drug conjugates are effective only againsttumors with very high ErbB2 expression levels.

Finally, the potency of anti-PRLR antibodies conjugated to DM1 via thenon-cleavable linker SMCC was compared to the cell killing potency ofanti-PRLR antibodies conjugated to MMAE via the cleavable linker:mc-vc-PAB (available from Concortis, San Diego, Calif.). Cells used inthis experiment were PC3, PC3/hPRLR, MCF7/ATCC and MCF7/PRLR. Resultsare shown in Table 18.

TABLE 18 Anti-PRLR ADC Cell Kill Potency PC3/ MCF7/ MCF7/ PC3 hPRLR ATCCPRLR IC₅₀ % IC₅₀ % IC₅₀ % IC₅₀ % (nM) Kill (nM) Kill (nM) Kill (nM) KillFree DM1 0.5 90 1.0 70 3.0 80 1.9 86 Free MMAE 1.4 90 1.0 77 2.4 83 1.595 Isotype Control I-SMCC- 95 79 100 79 100 71 100 76 DM1 IsotypeControl I-mc-VC- 200 33 145 55 143 23 143 21 PAB-MMAE H1H6953N-SMCC-DM190 81 0.4 83 80 70 0.1 80 H1H6953N-mc-VC-PAB- 130 49 0.2 80 150 42 0.185 MMAE H1H6958N2-SMCC-DM1 110 81 0.5 79 90 72 0.2 83 H1H6958N2-mc-VC-100 39 0.5 82 150 53 0.2 88 PAB-MMAE H1H6765P-SMCC-DM1 80 80 0.3 83 9071 0.1 81 H1H6765P-mc-VC-PAB- 150 29 0.40 85 150 38 0.2 86 MMAE

As shown in Table 18, nearly equivalent cell killing in PC3/hPRLR andMCF7/hPRLR cell lines was observed for both the non-cleavable DM1 ADCs(H1H6953-SMCC-DM1, H1H6958N2-SMCC-DM1, and H1H6765-SMCC-DM1) and for thecleavable MMAE ADCs (H1H6953-mc-vc-PAB-MMAE, H1H6958N2-mc-VC-PAB-MMAE,and H1H6765-mc-VC-PAB-MMAE).

Additional toxins (DM4, MeNHC3-May, and MMD) conjugated to anti-PRLRantibodies were also tested in T47D and MCF7/hPRLR cell lines, andresults are summarized in Tables 19 (293 and T47D cell killing) and 20(MCF7/ATCC and MCF7/PRLR cell killing). (ND=not detected).

TABLE 19 Anti-PRLR Antibody Drug Conjugates - Cell Killing Properties(293 and T47D Cell Lines) Cell Line 293 T47D IC₅₀ % IC₅₀ % AntibodyLinker Drug nM Kill nM Kill Free DM1 1.2 95 1.5 100 (Me-SS-May) MMAE 0.9100 1 100 DM4 0.6 100 0.5 100 MMD 0.9 100 2 100 MeNHC3-May 60 90 90 100Isotype SMCC DM1 150 80 140 50 Control I mc-VC-PAB MMAE 300 30 300 20MMD 300 70 140 70 MeNHC3-May 300 30 300 30 SPDB DM4 50 90 30 100H1H6953N SMCC DM1 100 90 1.5 100 mc-VC-PAB MMAE ND ND 1.0 80 MMD 110 701.0 90 MeNHC3-May 300 20 1.0 90 H1H6958N2 SMCC DM1 100 90 2 100mc-VC-PAB MMAE ND ND 1 90 SPDB DM4 ND ND 1 100 H1H6975P SMCC DM1 80 90 2100 mc-VC-PAB MMD 110 80 3 90 H1H6782P SMCC DM1 120 80 3 100 mc-VC-PABMMD 230 70 2 90 mc-VC-PAB MeNHC3-May 60 90 2 90 H1H6765P SMCC DM1 85 1001 90 mc-VC-PAB MMAE ND ND 1 90

TABLE 20 Anti-PRLR Antibody Drug Conjugates - Cell Killing Properties(MCF7/ATCC and MCF7/PRLR Cell Lines) Cell Line MCF7/ ATCC MCF7/PRLR IC₅₀% IC₅₀ % Antibody Linker Drug nM Kill nM Kill Free DM1 1.3 80 0.8 80(Me-SS-May) MMAE 2.4 80 1.5 95 DM4 1.3 90 2 90 MMD 2 90 0.4 90MeNHC3-May 100 70 50 80 Isotype SMCC DM1 300 60 150 70 Control Imc-VC-PAB MMAE 140 20 140 20 MMD 300 40 70 75 MeNHC3-May 300 30 200 55SPDB DM4 70 80 80 90 H1H6953N SMCC DM1 90 70 0.3 80 mc-VC-PAB MMAE 15040 0.1 85 MMD 300 50 0.2 90 MeNHC3-May 150 60 0.2 80 H1H6958N2 SMCC DM170 70 0.2 80 mc-VC-PAB MMAE 150 50 0.2 90 SPDB DM4 25 80 0.8 90 H1H6975PSMCC DM1 200 70 0.2 80 mc-VC-PAB MMD 150 50 0.2 90 H1H6782P SMCC DM1 25070 0.3 80 mc-VC-PAB MMD 200 50 0.3 90 mc-VC-PAB MeNHC3-May 250 50 0.2 80H1H6765P SMCC DM1 150 70 0.3 90 mc-VC-PAB MMAE 150 40 0.2 90

All tested anti PRLR ADCs, regardless of the toxin and linker utilized,specifically killed the tested cells. T47D cell viability IC₅₀s rangedfrom 0.6 nM to 3.4 nM and MCF7/hPRLR cell viability IC50s ranged from0.2 nM to 0.8 nM.

Example 8. Anti-PRLR Antibody-Drug Conjugates Effectively Inhibit TumorGrowth In Vivo

To determine the in vivo efficacy of the anti-PRLR-DM1 antibody-drugconjugates, studies were performed in immunocompromised mice bearingPRLR+ breast cancer xenografts.

Briefly, 20×10⁶ MCF7/PRLR cells (ATCC HTB-22 transfected with fulllength hPRLR as previously described) were implanted subcutaneously intothe left mammary fat pad of female NCr nude mice. In other studies,10×10⁶ PC3/PRLR (ATCC CRL-1435 transfected with full length hPRLR aspreviously described) were implanted subcutaneously into the left flankof male SCID mice. Additionally, 10×10⁶ parental T47D (ATCC HTB-133) or7.5×10¹⁰ T47D#11 cells (ATCC HTB-133 serially passaged in vivo asdescribed below) were subcutaneously implanted into the left flank offemale CB17 SCID mice. All mice were obtained from Taconic (Hudson,N.Y.). Each bolus of cells was supplemented with a 90-day estrogenrelease pellet (1.7 mg/pellet; Innovative Research America, SarasotaFla.). Once tumors had reached an average volume of 250 mm³, mice wererandomized into groups of seven and dosed with anti-PRLR antibody-drugconjugates or control reagents. Control reagents included PBS vehicle,free methyl-disulfide DM1 (DM1-SMe) and isotype Control 1-DM1.

In multi-dose studies, mice were dosed once a week for a total of threeweeks with tumor volumes and body weights being monitored twice weeklythroughout the study. Test ADCs were dosed at 5 and/or 15 mg/kg in themulti-dose studies. In single-dose studies, mice received a single doseof test ADC, and tumor volumes and body weights were monitored twiceweekly throughout the study. Test ADCs were dosed at 1, 2.5, 5, and 15mg/kg in the single-dose studies. Average tumor size as well as tumorgrowth inhibition relative to the vehicle treated group were calculatedfor each group. Tumors were measured with calipers twice a week untilthe average size of the vehicle group reached 1000 mm³. Tumor size wascalculated using the formula (length×width²)/2. Tumor growth inhibitionwas calculated according to the following formula:(1−((T_(final)−T_(initial))/(C_(final)−C_(initial))))*100, where T(treated group) and C (control group) represent the mean tumor mass onthe day the vehicle group reached 1000 mm³. Animals were observed to Day52. Results are summarized in Tables 21-25 (multi-dose) and Table 26(single dose). (NT=not tested in the particular experiment shown).

TABLE 21 Tumor Size and Tumor Growth Inhibition Following Multi-DoseAdministration of Anti-PRLR Antibody-Drug Conjugates and Controls -MCF7/PRLR tumors (TRIAL #1) [NCr Nude mice - data collected at Day 52]Average Tumor Dose Final Tumor size Growth Inhibition Treatment Group(mg/kg) mm³ (mean ± SEM) (%) Vehicle — 1068 ± 384  — Free DM1 0.2 625 ±141 57 Isotype control 5 NT NT Ab-DM1 15 300 ± 141 96 H1H6958N2 15 483 ±46  74 H1H6958N2-DM1 5 51 ± 33 128 15 0 ± 0 133 H1H6953N 15 421 ± 23  79H1H6953N-DM1 5 107 ± 45  120 15 0 ± 0 133 H1H6975N 15 659 ± 144 51H1H6975N-DM1 5 125 ± 46  118 15 0 ± 0 135 H1H6782P 15 NT NT H1H6782P-DM15 15 H1H6765P 15 NT NT H1H6765P-DM1 5 15

TABLE 22 Tumor Size and Tumor Growth Inhibition Following Multi-DoseAdministration of Anti-PRLR Antibody-Drug Conjugates and Controls -MCF7/PRLR tumors (TRIAL #2) [NCr Nude mice - data collected at Day 63]Average Tumor Dose Final Tumor size Growth Treatment Group (mg/kg) mm³(mean ± SEM) Inhibition (%) Vehicle — 870 ± 211 — Free DM1 0.2 1080 ±451  −33 Isotype control Ab-DM1 5 1106 ± 371  −39 15 712 ± 214 24H1H6958N2 15 766 ± 128 17 H1H6958N2-DM1 5 117 ± 10  116 15 0 ± 0 137H1H6953N 15 NT NT H1H6953N-DM1 5 15 H1H6975N 15 NT NT H1H6975N-DM1 5 15H1H6782P 15 300 ± 83  88 H1H6782P-DM1 5 74 ± 34 123 15 0 ± 0 137H1H6765P 15 737 ± 182 19 H1H6765P-DM1 5 90 ± 56 122 15 0 ± 0 136

TABLE 23 Tumor Size and Tumor Growth Inhibition Following Multi-DoseAdministration of Anti-PRLR Antibody-Drug Conjugates and Controls -PC3/PRLR tumors (TRIAL #1) [SCID mice - data collected at Day 63]Average Tumor Dose Final Tumor size Growth Treatment Group (mg/kg) mm³(mean ± SEM) Inhibition (%) Vehicle — 1311 ± 257 — Free DM1 0.2 1361 ±120 −5 Isotype control Ab-DM1 5 1379 ± 128 −7 15 1091 ± 93  19 H1H6958N215 1507 ± 106 −19 H1H6958N2-DM1 5 1247 ± 171 5 15 808 ± 83 46 H1H6953N15 1306 ± 127 0 H1H6953N-DM1 5 1058 ± 138 23 15 892 ± 53 39 H1H6975N 151185 ± 97  12 H1H6975N-DM1 5  973 ± 169 31 15 895 ± 63 38 H1H6782P 15 NTNT H1H6782P-DM1 5 15 H1H6765P 15 NT NT H1H6765P-DM1 5 15

TABLE 24 Tumor Size and Tumor Growth Inhibition Following Multi-DoseAdministration of Anti-PRLR Antibody-Drug Conjugates and Controls -PC3/PRLR tumors (TRIAL #2) [SCID mice - data collected at Day 55]Average Tumor Dose Final Tumor size Growth Treatment Group (mg/kg) mm³(mean ± SEM) Inhibition (%) Vehicle — 1222 ± 99  0 Free DM1 0.2 1147 ±59  7 Isotype control Ab-DM1 5 1052 ± 101 16 15 1049 ± 127 16 H1H6958N215  917 ± 253 28 H1H6958N2-DM1 5 566 ± 63 61 15 230 ± 22 94 H1H6953N 15NT NT H1H6953N-DM1 5 15 H1H6975N 15 NT NT H1H6975N-DM1 5 15 H1H6782P 151154 ± 212 6 H1H6782P-DM1 5 490 ± 63 69 15 321 ± 33 85 H1H6765P 15 1208± 72  1 H1H6765P-DM1 5 489 ± 70 70 15 181 ± 42 98

TABLE 25 Tumor Size and Tumor Growth Inhibition Following Multi-DoseAdministration of Anti-PRLR Antibody-Drug Conjugates and Controls -T47D#11 tumors [SCID mice - data collected at Day 66] Average Tumor DoseFinal Tumor size Growth Treatment Group (mg/kg) mm³ (mean ± SEM)Inhibition (%) Vehicle — 1234 ± 88  0 Free DM1 0.2 1433 ± 23  −19Isotype control Ab-DM1 5 1340 ± 176 −9 15 1678 ± 67  −42 H1H6958N2 151259 ± 122 −3 H1H6958N2-DM1 5 168 ± 19 102 15 44 ± 5 112 H1H6953N 15 NTNT H1H6953N-DM1 5 15 H1H6975N 15 NT NT H1H6975N-DM1 5 15 H1H6782P 151537 ± 111 −29 H1H6782P-DM1 5 293 ± 20 90 15 124 ± 36 106 H1H6765P 151278 ± 164 −3 H1H6765P-DM1 5 183 ± 28 100 15  69 ± 12 111

TABLE 26 Tumor Size and Tumor Growth Inhibition Following Single DoseAdministration of Anti-PRLR Antibody-Drug Conjugates and Controls -MCF7/PRLR tumors [data collected at Day 55] Average Tumor Dose FinalTumor size Growth Treatment Group (mg/kg) mm³ (mean ± SEM) Inhibition(%) Vehicle — 710 ± 249 0 Isotype Control Ab-DM1 15 514 ± 86  38H1H6958N2 15 703 ± 160 1 H1H6958N2-1 1 274 ± 142 86 H1H6958N2-1 2.5 172± 53  109 H1H6958N2-1 5 107 ± 26  120 H1H6958N2-1 15 33 ± 23 136

Discussion

In this example, five exemplary anti-PRLR antibodies conjugated to DM1were initially assessed for the ability to reduce MCF7/PRLR and PC3/PRLRtumor volume in multi-dose studies. In the first multi-dose trial (Table21), H1H6958N2-DM1, H1H6953N-DM1 and H1H6975N-DM1 antibodies potentlyinhibited MCF7/PRLR tumor growth at both 5 and 15 mg/kg doses. At thehighest dose, all three DM1 conjugated antibodies reduced tumors toundetectable levels, with a percent reduction in tumor volume of about133-135%. This finding was replicated in a second multi-dose trial(Table 22) when H1H6958N2-DM1 was tested alongside two additionalexemplary anti-PRLR antibodies conjugated to DM1: H1H6782P and H1H6765P.In this second trial, tumor growth was also reduced to undetectablelevels at the highest dose of 15 mg/kg, with percent reduction in tumorvolume of 136-137%. Although treatment with unconjugated anti-PRLRantibodies resulted in moderate reduction of tumor volume (17-79%)compared to the vehicle group, the greatest inhibition in tumor size wasobserved in cohorts treated with antibody-drug conjugates.

Next, the anti-tumor efficacy of these same exemplary anti-PRLR-DM1antibodies was assessed in multi-dose studies in mice bearing PRLRpositive PC3/PRLR xenografts. (Tables 23 and 24). Mice were treatedafter tumors had grown for 21 days. H1H6958N2-DM1, H1H6953N-DM1 andH1H6975N-DM1 all demonstrated inhibition of tumor growth, especially atthe highest dose of 15 mg/kg. (Table 23). Anti-tumor effect wassimilarly observed in a second trial, when H1H6958N2-DM1 was testedalongside H1H6765P-DM1 and H1H6782P-DM1 after 15 days of tumor growth.At the highest dose administered, tumor inhibition across trials rangedfrom 38-98%. (Table 24). In comparison, an Isotype-control conjugated toDM1 produced only 16% tumor inhibition with final tumor volumes notsignificantly different to vehicle controls.

A further assessment of the anti-PRLR ADCs repeatedly dosed at 5 and 15mg/kg was performed in mice bearing T47D#11 xenografts endogenouslyexpressing PRLR. (Table 25). As in other tumor models, dosing wasinitiated when tumor size averaged 200 mm³. Results obtained in thistumor model were consistent with earlier results and clearlydemonstrated the anti-tumor activity of the anti-PRLR antibodiesconjugated to DM1. For example, H1H6958N2-DM1, H1H6765P-DM1 andH1H6782P-DM1 ADCs potently inhibited tumor growth at both the 5 and 15mg/kg dose. At 5 mg/kg anti-PRLR-DM1 conjugated antibodies exhibited89-100% tumor inhibition whereas at 15 mg/kg DM1-conjugated antibodiesresulted in 106-112% tumor growth inhibition. Importantly, unconjugatedanti-PRLR antibodies were not observed to have any anti-tumor efficacyin this endogenous tumor model, indicating the role of the DM1 conjugatein producing anti-tumor efficacy. Again, efficacy of anti-PRLR ADC wasvery specific as control ADC failed to have any effect on tumor growth.

In a final example, anti-PRLR DM1-conjugated antibody H1H6958N2 wasassessed in MCF7/PRLR xenografted mice in a single-dose study. (Table26). As in multiple-dose studies, established tumors were allowed togrow to approximately 200 mm³ before a single dose was administered.Here, H1H6958N2-DM1 was given at 1, 2.5, 5 and 15 mg/kg. As summarizedin Table 26, a dose dependent anti-tumor effect was seen across the widerange used in this study. Anti-tumor effect was observed at all doses,with 1 mg/kg causing a significant decrease in tumor volume relative tovehicle control tumors. Further, although 15 mg/kg of Isotype ControlI-DM1 had some anti-tumor effect (˜38% tumor growth inhibition), dosesof anti-PRLR-DM1 at 2.5 mg/kg and higher significantly reduced tumorvolume (>100% tumor growth inhibition at all doses above 1 mg/kgtested). Single doses of 5 and 15 mg/kg demonstrated anti-tumor efficacycomparable to that observed following repeat dosing at the same level,illustrating the potency of the anti-PRLR ADCs.

In summary, this example illustrates that conjugated anti-PRLRantibodies are potent inhibitors of tumor growth and are able to reducetumor size to undetectable levels in the various tumor models tested.

Example 9. Antibody-Drug Conjugates Against Class-I Cytokine ReceptorsEffectively Kill Cell Lines Expressing Low Levels of Target Antigen

As discussed elsewhere herein, antibody-drug conjugates against PRLReffectively kill PRLR-expressing cell lines, even those that expressrelatively low levels of target antigen. As previously noted, PRLRbelongs to the class I cytokine receptor family, which includes IL-4Rand IL-6R. Similar to PRLR, IL-4R and IL-6R are single-passtransmembrane receptors; IL-4R mediates IL-4 and IL-13 signaling, whileIL-6R mediates IL-6 signaling via a co-complex with the gp130 receptor.In further support of the general concept that ADCs directed againstclass I cytokine receptors may be used to effectively kill cells,including cells that express low-levels of target antigen, thecell-killing ability of ADCs directed against IL-4R and IL-6R wasevaluated.

Cell surface antigen levels on cells that endogenously or recombinantlyexpress IL-4R or IL-6R were first established using FACS. Briefly,approximately 1 million KG-1 (IL-4R⁺), HEK293/IL-4R and Ramos (IL-6R⁺)cells were incubated with exemplary anti-IL-4R (H4H083P2, see U.S. Pat.No. 7,608,693) and anti-IL-6R (VV6A9-5, see U.S. Pat. No. 7,582,298)antibodies for 30 min on ice. After washing, a PE-conjugated anti-humansecondary antibody (10 μg/ml) was added for 30 min followed by a secondwashing step and subsequent analysis on an Accuri C6 cytometer usingFlowJo software (Tree Star, Inc., Ashland, Oreg.). Relative IL-4R andIL-6R cell surface expression levels were calculated as the meanfluorescence intensity (MFI) above isotype control levels. Expressionlevels are summarized in Table 27.

TABLE 27 Relative IL-4R and IL-6R Cell Surface Expression on IL-4R andIL-6R Endogenously or Recombinantly Expressing Cell Lines Cell LineExpression Level; Fold Over Background Receptor HEK293/ ExpressionHEK293 IL4R KG-1 Ramos IL-4R 2 50 1 4 IL-6R 1 1 7 1

As shown in Table 27, HEK293 and Ramos cells endogenously expressedIL-4R at levels 2-fold and 4-fold over background, respectively, whilethe engineered HEK293/IL-4R cell line expressed IL-4R at levels 50-foldabove background. IL-4R expression was undetectable over background onKG-1 cells. IL-6R expression was detected at 7-fold above backgroundlevels in KG-1 cells, and not on HEK293 or Ramos cell lines.

Next, exemplary anti-IL-4R (H4H083P2) and anti-IL-6R (VV6A9-5)antibodies were conjugated to the cytotoxic drug DM1 and their potencyin cytotoxicity assays was evaluated. Briefly, HEK293/IL-4R, Ramos orKG-1 cell lines, as well as HEK293 parental cells were seeded inPDL-coated 96-well plates at 1500 to 10,000 cells per well. ADCs or freeDM1 (as the methyl disulfide derivative DM1-SMe) were added to the cellsat final concentrations ranging from 300 nM to 15 pM and incubated for 3days. Cells were incubated with CCK8 (Dojindo, Rockville, Md.) for thefinal 1-3 hours and the absorbance at 450 nm (OD₄₅₀) was determined on aFlexstation3 (Molecular Devices, Sunnyvale, Calif.). Background OD₄₅₀values from digitonin (40 nM) treated cells were subtracted from allwells and viability was expressed as a percentage of the untreatedcontrols. IC₅₀ values were determined from a four-parameter logisticequation over a 10-point response curve (GraphPad Prism). Results arepresented in Table 28.

TABLE 28 Cell killing Properties of Anti-IL4R and Anti-IL6R AntibodyDrug Conjugates on IL4R and IL-6R-Expressing Cell Lines HEK293/ HEK293hIL4R KG-1 Ramos Antibody-Drug IC₅₀ % IC₅₀ % IC₅₀ % IC₅₀ % Conjugate(nM) Kill (nM) Kill (nM) Kill (nM) Kill DM1 (Free Drug) 0.27 100 0.33 981.26 89 0.34 100 Isotype control-DM1 70 89 110 91 100 74 60 91Anti-IL-4R-DM1 80 91 0.22 95 90 83 18 91 Anti-IL-6R-DM1 100 88 70 92 3877 70 94 Receptor Expression Levels: Fold over Isotype Ctrl IL-4R 2 50 14 IL-6R 1 1 7 1

As shown in Table 28, anti-IL-4R-DM1 antibody-drug conjugates reducedRamos cell viability with an IC₅₀ value of 18 nM despite an IL-4Rsurface expression of only 4-fold above background levels. IL-4R-DM1ADCs reduced high IL-4R-expressing HEK293/IL-4R viability with an IC₅₀of 0.22 nM. Anti-IL-6R-DM1 antibody-drug conjugates had a modest butreproducible impact on the viability of KG-1 cells (expressing IL-6R ata level of 7-fold above background) with an IC₅₀ value of 38 nM comparedto an IC₅₀ value of 100 nM by an equivalently conjugated isotype controlADC.

In summary, this Example demonstrates that anti-IL-4R and anti-IL-6Rantibody drug conjugates exhibited potent and reproducible cytotoxicityeven on cell lines expressing modest receptor levels. This result issimilar to what was observed with anti-PRLR ADCs where potent cellkilling was obtained even on cells expressing low levels of PRLR (see,e.g., Example 7 herein). Thus, this Example provides further support forthe inventive concept that anti-class I cytokine receptor ADCs ingeneral may be effective therapeutic agents against cell lines andtumors that express class I cytokine receptors even at low levels.

Example 10. Epitope Mapping by Hydrogen/Deuterium Exchange

Experiments were conducted to determine the amino acid residues of PRLRwith which H1H6958N2 interacts. For this purpose hydrogen/deuterium(H/D) exchange epitope mapping was carried out. A general description ofthe H/D exchange method is set forth in e.g., Ehring (1999) AnalyticalBiochemistry 267(2):252-259; and Engen and Smith (2001) Anal. Chem.73:256A-265A.

To map the binding epitope(s) of antibody H1H6958N2 on hPRLR via H/Dexchange, a construct comprising a recombinant extracellular domain ofhPRLR with a C-terminal myc-myc-hexahistidine tag (hPRLR-mmH; SEQ ID NO:SEQ ID NO:406) was buffer exchanged to PBS with a final concentration of8 mg/mL, and antibody H1H6958N2 was covalently attached toN-hydroxysuccinimide (NHS) agarose beads (GE Lifescience)

In the ‘on-solution/off-beads’ (on-exchange in solution followed byoff-exchange on beads) experiment, the hPRLR was deuterated for 5 min or10 min in PBS buffer prepared with D₂O, and then bound to H1H6958N2beads through a 2 min incubation. The hPRLR-bound beads were washed withPBS aqueous buffer (prepared with H₂O) and incubated PBS buffer for halfof the on-exchange time. After the off-exchange, the bound hPRLR waseluted from beads with an ice-cold low pH TFA solution. The eluted hPRLRwas then digested with immobilized pepsin (Thermo Scientific) for 5 min.The resulting peptides were desalted using ZipTip® chromatographicpipette tips and immediately analyzed by UltrafleXtreme matrix assistedlaser desorption ionization time of flight (MALDI-TOF)-TOF massspectrometry (MS).

In the ‘on-beads/off-beads’ (on-exchange on beads followed byoff-exchange on beads) experiment, hPRLR was first bound to H1H6958N2beads and then incubated for 5 min or 10 min in D₂O for on-exchange. Thefollowing steps (off-exchange, pepsin digestion, and MS analysis) werecarried out as described for the ‘on-solution/off-beads’ procedure. Thecentroid values or average mass-to-charge ratios (m/z) of all thedetected peptides were calculated and compared between these two sets ofexperiments.

The results are summarized in Table 29 which provides a comparison ofthe centroid m/z values for all the detected peptides identified byliquid chromatography and MS following the H/D exchange and pepticdigest procedure described above. For purposes of the present Example, apositive difference (A) of at least 0.20 m/z in both experimentsindicates amino acids protected by antibody binding. Segments meetingthis criterion are indicated by bold text and an asterisk (*) in Table29. [Note that, although this experiment was conducted with a truncatedform of PRLR (i.e., hPRLR-mmH; SEQ ID NO: 406), residue numbering in thefar left column of Table 29 has been adjusted to correspond to the aminoacid sequence of the full-length PRLR protein represented by SEQ ID NO:404.]

TABLE 29 H1H6958N2 Binding to hPRLR-mmH Experiment I 5 min Experiment II10 min Residues on-/2.5 min off-exchange on-/5 min off-exchange (of SEQon- on- ID solution/ on-beads/ solution/ on-beads/ NO: 404) off beadsoff-beads Δ off beads off-beads Δ 61-71 1306.39 1306.50 −0.10 1306.341306.38 −0.04 64-71 1005.13 1005.05 0.07 1005.13 1005.18 −0.05 64-721136.16 1136.15 0.01 1136.33 1136.49 −0.16  72-94* 2573.91 2572.17 1.742573.74 2572.12 1.62  72-95* 2705.49 2703.43 2.06 2705.29 2703.70 1.60 96-101* 870.70 870.20 0.50 870.71 870.20 0.52  96-102* 1001.59 1001.220.37 1001.62 1001.14 0.48 133-147 1786.59 1786.37 0.22 1786.55 1786.520.02 134-147 1673.36 1673.29 0.07 1673.50 1673.39 0.11 135-147 1602.151602.02 0.13 1602.15 1602.04 0.11 136-147 1502.85 1502.88 −0.03 1502.791502.88 −0.09 137-147 1373.78 1373.68 0.09 1373.80 1373.84 −0.04 168-1791447.73 1447.71 0.02 1447.76 1447.78 −0.02 168-180 1633.59 1633.66 −0.061633.71 1633.64 0.08 169-180 1469.67 1469.86 −0.19 1469.71 1469.67 0.04170-179 1155.40 1155.35 0.05 1155.40 1155.39 0.01 170-180 1341.621341.66 −0.04 1341.49 1341.34 0.15 191-205 1772.03 1772.03 −0.01 1772.151772.28 −0.13 192-202 1284.36 1284.39 −0.03 1284.32 1284.43 −0.11192-205 1624.94 1624.98 −0.04 1625.08 1625.17 −0.10 206-215 1261.441261.33 0.11 1261.24 1261.46 −0.22 206-217 1419.64 1419.65 −0.01 1419.481419.45 0.02 206-222 1962.58 1962.39 0.20 1962.52 1962.63 −0.10 236-2491560.81 1560.76 0.05 1560.81 1560.83 −0.01 238-261 2828.15 2827.99 0.162827.99 2828.17 −0.18 241-261 2498.70 2498.74 −0.04 2498.68 2498.88−0.20 250-261 1528.68 1528.75 −0.06 1528.64 1528.71 −0.07

While the majority of the observed peptic peptides gave similar centroidvalues for both the on-solution/off-beads and on-beads/off-beadsprotocols, the region corresponding to amino acid residues 72 to 102 ofSEQ ID NO: 404 had delta centroid values greater than 0.20 m/z in bothexperiments. The H/D exchange results summarized in Table 29 thereforeindicate that the region corresponding to amino acids 72 to 102 of SEQID NO: 404 (i.e., MHECPDYITGGPNSCHFGKQYTSMWRTYIMM, SEQ ID: 405)comprises the epitope of PRLR with which antibody H1H6958N2 interacts.This epitope is located within the fibronectin-like type III domain 1(“Domain 1”) of the extracellular domain of PRLR. Thus, the presentExample, in conjunction with other working examples set forth herein,identifies Domain 1 of the PRLR extracellular domain as a particularlyeffective target for antibodies and ADCs that are useful for treatingPRLR-related diseases and disorders.

Example 11. PRLR Expression on Cell Line Surfaces and in Human BreastCancers

To assess the expression of PRLR in human breast cancer, samples ofbreast cancer tumors were assessed using RNAscope In Situ Hybridizationtechnology (Advanced Cell Diagnostics). FFPE embedded samples were fromBreast Tumor Micro-Arrays (TMAs) obtained from Indivumed. Tumor sectionswere assessed for PRLR RNA expression using specific RNA Scope probesand developed using Diaminobenzidine chromogenic stains according tostandard protocols. Images were scanned on an Aperio XT Scan scope at40× (Leica Systems) and PRLR expression was assessed in the samplesqualitatively.

PRLR expression was heterogeneous across some tumors (data not shown).No correlation between PRLR expression and hormone receptor or HER2status of the tumors was observed, with PRLR found in both hormonereceptor positive tumors and ER/PR/HER2 “triple negative” tumors. Thesedata are consistent with analysis of PRLR mRNA levels in human breastcancer from public gene expression databases (data not shown).

Example 12. Anti-PRLR ADCs Block Binding and PRL Induced STAT5 Activity

ELISA-based blocking assays were developed to determine the ability ofH1H6958N2 and H1H6958N2-DM1 to block binding of human and cynomolgusmonkey PRLR to immobilized human PRL. Dimeric forms of human orcynomolgus monkey (Macaca fascicularis) PRLR and monomeric human PRLwere used in this assay. To generate initial dose-response curves, humanor monkey PRLR at concentrations ranging from 3.38 pM to 200 nM wasadded to the plates previously coated in 1 μg/mL PRL. After 1-hourincubation, captured PRLR was detected with HRP-conjugated goat anti-Fcγantibody and visualized with TMB colorimetric substrate by measuringabsorbance at 450 nm. To determine the inhibition of PRLR binding byH1H6958N2 or H1H6958N2-DM1, 10 nM of human or monkey PRLR was mixed withmAbs or ADC ranging from 520 pM to 30 nM and incubated at roomtemperature for 1 hour. Mixtures were then added to plates coated within 1 μg/mL PRL and captured PRLR was detected as described above. Datawere analyzed using a four-parameter logistic equation over an 11-pointresponse curve in GraphPad Prism software and IC₅₀ values werecalculated.

The capacity of H1H6958N2 and H1H6958N2-DM1 to block human PRL-inducedPRLR signaling was determined in a luciferase reporter assay using theHEK293 cell line engineered to constitutively express PRLR and stablytransduced with a lucifierase reporter construct comprising 5 tandemSTAT5 binding sites derived from the promoter of the human interleukin-2receptor α gene [STAT5 response element (×5)-luciferase] (John et al.,The EMBO Journal, 1996, 15(20): 5627-5635). STAT5 transcription factorsare known mediators of intracellular signal transduction downstream ofPRLR (Fang et al., BMC Biotechnology, 2008, 8:11. To generate adose-response curve, human PRL was added to the reporter cells atconcentrations ranging from 0.8 pM to 100 nM. Following addition of PRL,cells were incubated for 6 hours at 37° C. and 5% CO₂ and thenequilibrated to room temperature for 15 minutes. Luciferase activity wasmeasured using the ONE-Glo substrate and measuring luminescence. To testfor inhibition of PRL-mediated PRLR signaling in the cells, mAbs or ADCswere added at concentrations ranging from 3.3 pM to 200 nM (molarityindicates antibody concentration for both ADC and mAb), followed by theaddition of a constant concentration of 2 nM PRL. Plates were incubatedat 37° C. and 5% CO₂ for 5 hours and equilibrated to room temperaturefor 10 minutes. Luciferase activity was again measured using the ONE-Glosubstrate and measuring luminescence.

H1H6958N2-DM1 blocked the binding of human or monkey PRLR to monomerichuman PRL ligand by ELISA (FIG. 1). Here, human or monkey PRLR bound tohuman PRL in a dose-dependent manner, with EC₅₀ values of 4.9 and 5.7nM, respectively (solid black squares in insets in FIGS. 1A and 1B).H1H6958N2-DM1 and H1H6958N2 blocked the binding of 10 nM human PRLR toimmobilized human PRL with IC₅₀ values of 4.4 nM and 5.0 nM,respectively (FIG. 1A). In a similar manner, H1H6958N2-DM1 and H1H6958N2blocked the binding of monkey PRLR to human PRL with IC₅₀ values of 4.2nM and 5.8 nM, respectively (FIG. 1B). Control antibody and control ADCdid not demonstrate any blocking activity under identical assayconditions.

To ensure that H1H6958N2-DM1 also blocked PRL induced STAT5 activity, aHEK293/PRLR/STAT5-Luc reporter cell line was generated. In this reporterline, 2.1 nM of human PRL was determined to be the concentrationrequired for stimulation of human PRLR signaling to 50% of the maximumactivity level (EC₅₀) (FIG. 1C). Both H1H6958N2 and H1H6958N2-DM1effectively blocked PLR-induced luciferase activity in a dose-dependentmanner, with 0.4 nM of H1H6958N2 and H1H6958N2-DM1 required for a 50%reduction of PRLR signaling (IC₅₀) in the presence of a constantconcentration of 2 nM PRL. As with the blocking ELISA assays, neithercontrol mAb nor control ADC blocked PRL-induced PRLR-STAT5 signaling.

In summary, in addition to the cytotoxic activity of DM1, H1H6958N2-DM1effectively blocks PRL binding to PRLR and blocks PRL-induced STAT5signaling.

Example 13. H1H6958N2-DM1 has Anti-Tumor Activity Against MCF7 andMCF7/PRLR Breast Cancer Xenografts

Additional experiments were performed to assessing the overallanti-tumor activity of H1H6958N2-DM1 in several breast tumor xenograftmodels expressing both endogenous and transfected PRLR.

After MCF7 or MCF7/PRLR tumors reached an average volume of ˜150-200 mm³(14-16 days post implantation), mice were randomized into treatmentgroups (n=8-9 mice/group) and received a single dose or threeonce-weekly doses of H1H6958N2-DM1, control ADC, or vehicle control,administered systemically by intravenous tail-vein injection. Tumor sizeand body weight were measured in vivo twice per week throughout theduration of the study.

Activity of H1H6958N2-DM1 in a tumor expressing low levels of thereceptor was assessed using NCr nude mice bearing established MCF7 tumorxenografts. Although this tumor cell line was not inhibited byH1H6958N2-DM1 in vitro (see above), moderate in vivo anti-tumor activitywas observed following a single dose of 5 or 10 mg/kg H1H6958N2-DM1(FIG. 2A). At the highest dose assessed, (15 mg/kg), H1H6958N2-DM1significantly inhibited the growth of the MCF7 tumors relative tocontrol ADC. Repeat treatment of MCF7 tumors in a subsequent studyresulted in more pronounced anti-tumor effects than observed in thesingle dose study. Here, both 10 and 15 mg/kg H1H6958N2-DM1significantly inhibited MCF7 tumor growth relative to control ADC,although some anti-tumor activity was induced by control ADC relative tovehicle control (FIG. 2B).

The therapeutic efficacy of H1H6958N2-DM1 was next assessed in theMCF7/PRLR tumor xenograft expressing high levels of PRLR. Here,treatment with a single dose of H1H6958N2-DM1 at 2.5-15 mg/kg wasactive, with significant antitumor efficacy observed at all doses.Increased activity at 10 and 15 mg/kg was observed, with these dosescausing eradication of the tumors over the course of the study (FIG.2C). Administration of three once-weekly doses demonstrated furthersignificant anti-tumor activity, with repeat doses of 5 and 15 mg/kgH1H6958N2-DM1 leading to tumor eradication in this model (FIG. 2D). Noanti-tumor activity was observed for control ADC in this model andunconjugated H1H6958N2 that was assessed in this study also failed toinduce any inhibition of tumor growth.

MCF7 xenografts showed a clear response to H1H6958N2-DM1 in vivo despiteexpressing moderate levels of the PRLR and being insensitive to ADC invitro. Greater exposure to H1H6958N2-DM1 in vivo, or a relative increasein PRLR expression in vivo may be responsible for the differentialresponse observed.

Example 14: H1H6958N2-DM1 Anti-Tumor Activity Against T47DvII BreastCancer Xenografts and Combination with Fulvestrant Increases Activity

The T47Dv11 model was used to assess the activity of H1H6958N2-DM1 in amodel with significant levels of endogenous PRLR. As parental T47D cellsare poorly tumorigenic, in vivo passaging was employed to develop avariant with consistent tumorigenicity. Initially, 10×10⁶ parental T47Dcells were implanted subcutaneously (SC) into the left flank of femaleC.B.-17 SCID mice (Taconic, Hudson N.Y.). Tumors were supplemented witha 90-day release 1.7 mg estrogen pellet (Innovative Research America).Where T47D tumors were observed, they were excised and cut into 3 mmfragments and subsequently implanted into the left flank of separatefemale C.B.-17 SCID mice. Subsequent passaging was performed until alarge tumor was disaggregated into a single cell suspension. The cells(named T47Dv11) from this tumor were then cultured in vitro andexpanded. Flow cytometry confirmed that generation of T47DvII cells didnot alter PRLR expression relative to the parental cell (data notshown). For efficacy studies, mice were implanted with 7.5×10⁶ T47Dv11cells into the left flank of female C.B.-17 SCID mice supplemented witha 90-day release 0.72 mg/pellet estrogen pellet. For MCF7 and MCF7/PRLRtumor xenografts, 2×10⁷ MCF7/PRLR cells were implanted SC into the leftmammary fat pad of female NCr nude mice (7-8 weeks old) supplementedwith 90-day release 0.72 mg/pellet estrogen pellets. Tumors weremeasured with calipers twice a week for the duration of each study.Tumor volume was calculated using the formula TV=(length×width²)/2.

After T47Dv11 tumors reached an average volume of ˜150-200 mm³ (14-16days post implantation), mice were randomized into treatment groups(n=8-9 mice/group) and received a single dose or three once-weekly dosesof H1H6958N2-DM1, control ADC, or vehicle control, administeredsystemically by intravenous tail-vein injection. The selective estrogenreceptor down-regulator (SERD) fulvestrant (Faslodex, Astra Zeneca) wasadministered subcutaneously in its recommended vehicle (10% w/v Alcohol,10% w/v Benzyl Alcohol, and 15% w/v Benzyl Benzoate, made up to 100% w/vwith Castor Oil) once weekly for the duration of studies. Tumor size andbody weight were measured in vivo twice per week throughout the durationof the study.

Single dose treatment with H1H6958N2-DM1 at either 2.5 or 5 mg/kgsignificantly inhibited tumor growth (FIG. 3A). As observed in the othermodels, repeat dosing induced more robust anti-tumor efficacy; threedoses of either 5 or 15 mg/kg H1H6958N2-DM1 caused tumor regression.Complete regressions were apparent for some tumors that received thehighest dose of ADC (FIG. 3B). No activity of control ADC was apparentin the T47Dv11 model, and unconjugated H1H6958N2 again had no effect ontumor growth.

Next, the combination of H1H6958N2-DM1 with the SERD fulvestrant wastested in the T47Dv11 xenograft model. Here, the activity of eitheragent at partially effective doses was compared against the activity ofthe agents in combination. First, a dose titration study of weeklyadministration of fulvestrant was performed in order to establish itssingle agent activity (FIG. 4). Separate in vitro experimentsdemonstrated that continuous culture of T47DvII cells with cytostaticconcentrations of fulvestrant (10 or 100 nM) did not significantlyreduce PRLR expression as assessed by flow cytometry or western blotting(data not shown). Significant anti-tumor activity was observed followingthe individual treatments of 2.5 mg/kg H1H6958N2-DM1 and 150 or 250mg/kg fulvestrant. Combination of these agents was observed to cause asignificant increase in anti-tumor activity, providing further evidencethat the combination of H1H6958N2-DM1 with fulvestrant has additiveefficacy in ER positive tumors (FIG. 3C) and Table 30.

TABLE 30 Inhibition of T47DvII Tumor Growth at Day 70 in SCID micetreated with anti-PRLR antibody-drug conjugates and fulvestrant FinalTumor Average Tumor Volume Growth Treatment Group (mean ± SD) Inhibition(%) PBS Vehicle 1212v ± 95   0 H1H6958N2-DM1 2.5 mg/kg 386 ± 249 77Isotype Control Ab-DM1 2.5 mg/kg 1177 ± 229  3 Fulvestrant 150 mg/kg 660± 205 52 Fulvestrant 250 mg/kg 433 ± 189 74 Isotype Control Ab-DM1 + 758± 360 42 Fulvestrant 150/mg/kg Isotype Control Ab-DM1 + 552 ± 282 62Fulvestrant 250/mg/kg H1H6958N2-DM1 + Fulvestrant 148 ± 81  101150/mg/kg H1H6958N2-DM1 + Fulvestrant 150 ± 74  100 250/mg/kg

Measurement of Tumor Growth and Inhibition

For the subcutaneous tumors, average tumor size as well as tumor growthinhibition relative to the Vehicle treated group were calculated foreach group. Tumors were measured with calipers twice a week until theaverage size of the vehicle group reached 1200 mm³. Tumor size wascalculated using the formula (length×width²)/2. For all studies, tumorgrowth inhibition was calculated according to the following formula:(1−((T_(final)−T_(initial))/(C_(final)−C_(initial))))*100, where T(treated group) and C (control group) represent the mean tumor mass onthe day the vehicle group reached 1000 mm³.

In summary, combined with surprising preliminary in vitro experimentsthat demonstrated that culture of cells with cytostatic concentrationsof fulvestrant did not exhibit significantly reduced PRLR expression,these in vivo results demonstrate that H1H6958N2-DM1 in combination withfulvestrant had superior anti-tumor efficacy relative to the singleagents and the combination is useful in treating PRLR positive breastcancer.

Example 15. Pharmacodynamic Assessment of H1H6958N2-DM1 Activity In Vivo

In order to validate the activity of DM1 on microtubule activity, theinduction of mitotic arrest following treatment of T47DvII tumors withH1H6958N2-DM1 in vivo was assessed by phospho-histone-H3 (pHH3) IHC.Mice were implanted with T47DvII cells and tumors were grown for 15 dayswhere mice were randomized based on tumor volume and dosed intravenouslywith either vehicle, H1H6958N2-DM1 at 5 or 15 mg/kg or Control ADC at 15mg/kg (N=4-5). At 24, 48 or 72 hours post-treatment, mice wereeuthanized and tumors processed as FFPE samples for IHC analysis. Tumorsections were then stained with anti-phospho Histone H3 (pHH3) antibody(clone: Ser10, Cell Signaling Technology #9701) as a measure of mitoticcells. Images were scanned on an Aperio XT Scan scope at 40× (LeicaSystems). PHH3 positive nuclear signals were imaged and quantified withthe Halo CytoNuclear Count algorithm (Halo, Indica Labs). The percent ofpositive cells in each tumor was quantified for each group and anaverage calculated for each treatment condition.

Tumors treated at a dose of 5 mg/kg H1H6958N2-DM1 showed clear inductionof pHH3 immunoreactivity, with a significant increase relative tovehicle treated tumors (FIG. 5). At a higher H1H6958N2-DM1 dose of 15mg/kg, a further increase in pHH3 levels in tumors was apparent (datanot shown). Surprisingly, levels of pHH3 peaked at 48 hours posttreatment and were somewhat lower at 72 hours, suggesting the time ofmaximal activity of DM1 on microtubules and cell cycle arrest.Importantly, the induction of pHH3 in tumors was specific to treatmentwith H1H6958N2-DM1, as tumors treated with 15 mg/kg control ADC did nothave a significant increase in pHH3 over vehicle treated tumors.Accordingly, levels of pHH3 expression resulting from treatment witheither 5 or 15 mg/kg H1H6958N2-DM1 were significantly higher than thatobserved in control ADC treated tumors.

In summary, the targeted delivery of DM1 following specificinternalization of H1H6958N2-DM1 increased levels of pHH3 in treatedtumors, indicative of mitotic arrest.

Example 16: H1H6958N2-DM1 Anti-Tumor Activity Against TM00107 PatientDerived Xenograft Tumor

Efficacy of H1H6958N2-DM1 was also assessed in a patient derivedxenograft (PDX) model of breast cancer that may better represent theresponse in patients. NOD SCID gamma (NSG) mice bearing tumor fragmentsof the TM00107 PDX breast cancer tumor were sourced from The JacksonLaboratories. The TM00107 PDX model was derived from an ER+/−/PR−/HER2−metastatic breast adenocarcinoma. PRLR expression was confirmed bymicroarray analysis and immunohistochemistry on tumor sample slidesprovided by The Jackson Laboratory (data not shown). When tumors were anaverage volume of ˜400-600 mm³ (21 days post implantation), mice wererandomized into treatment groups (n=7 mice/group) and were administeredH1H6958N2-DM1, control ADC, or vehicle control by intravenous injectiononce weekly for a total of 4 weeks. A separate treatment group wasadministered paclitaxel by intravenous injection every 4 days for atotal of 4 doses.

Control tumors grew very rapidly, and mice that received vehicle controlwere euthanized at day 30 post-implantation due to large tumor volume.At this time, a moderate but significant (p<0.01) decrease in averagetumor volumes of mice treated with 20 mg/kg H1H6958N2-DM1 was observedcompared to animals administered 20 mg/kg control ADC (FIG. 6A). A trendtowards inhibition of tumor growth was observed in mice treated with thelower dose of 10 mg/kg H1H6958N2-DM1 compared to vehicle control mice,however, no significant difference in average tumor volume was observed.The paclitaxel treatment did not have an effect on tumor growth.

These experiments demonstrate the usefulness of H1H6958N2-DM1 intreating PRLR positive breast cancer in a PDX model of breast cancer.

The present disclosure is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications in additionto those described herein will become apparent to those skilled in theart from the foregoing description and the accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims.

1. A method of treating PRLR positive breast cancer, the methodcomprising co-administering to a patient a therapeutically effectiveamount of: (a) an antibody-drug conjugate (ADC) comprising an antibodyor antigen-binding fragment thereof conjugated to a maytansinoid,wherein the antibody or antigen-binding fragment thereof binds to humanprolactin receptor (PRLR), and (b) fulvestrant.
 2. The method of claim1, wherein the antibody blocks prolactin mediated signaling in cellsexpressing PRLR with an IC50 of less than about 1.3 nM.
 3. The method ofclaim 1, wherein the antibody blocks prolactin mediated signaling incells expressing PRLR with an IC50 of less than about 1.2 nM.
 4. Themethod of claim 1, wherein the antibody blocks prolactin mediatedsignaling in cells expressing PRLR with an IC50 of less than about 1.0nM.
 5. The method of claim 1, wherein the antibody blocks prolactinmediated signaling in cells expressing PRLR with an IC50 of less thanabout 900 pM.
 6. The method of claim 1, wherein the antibody blocksprolactin mediated signaling in cells expressing PRLR with an IC50 ofless than about 800 pM.
 7. The method of claim 1, wherein the antibodyblocks prolactin mediated signaling in cells expressing PRLR with anIC50 of less than about 600 pM.
 8. The method of claim 1, wherein theantibody blocks prolactin mediated signaling in cells expressing PRLRwith an IC50 of less than about 400 pM.
 9. The method of claim 1,wherein the antibody blocks prolactin mediated signaling in cellsexpressing PRLR with an IC50 of less than about 200 pM.
 10. The methodof claim 1, wherein the antibody blocks prolactin mediated signaling incells expressing PRLR with an IC50 of less than about 100 pM.
 11. Themethod of claim 1, wherein the antibody blocks prolactin mediatedsignaling in cells expressing PRLR with an IC50 of less than about 80pM.
 12. The method of claim 1, wherein the antibody blocks prolactinmediated signaling in cells expressing PRLR with an IC50 of less thanabout 60 pM.
 13. The method of claim 1, wherein the antibody blocksprolactin mediated signaling in cells expressing PRLR with an IC50 ofless than about 40 pM.
 14. The method of claim 1, wherein the antibodyor antigen-binding fragment thereof that binds prolactin receptor (PRLR)interacts with one or more amino acids contained within the firstfibronectin-like type III domain of the extracellular domain of PRLR(amino acids 27-128 of SEQ ID NO:404), as determined byhydrogen/deuterium exchange.
 15. The method of claim 14, wherein theantibody or antigen-binding fragment thereof interacts with one or moreamino acids contained within SEQ ID NO:405, as determined byhydrogen/deuterium exchange.
 16. The method of claim 14, wherein theantibody or antigen-binding fragment thereof interacts with an aminoacid sequence selected from the group consisting of (a) amino acids 72to 94 of SEQ ID NO:404; (b) amino acids 72 to 95 of SEQ ID NO:404; (c)amino acids 96 to 101 of SEQ ID NO:404; and (d) amino acids 96 to 102 ofSEQ ID NO:404.
 17. The method of claim 15, wherein the antibody orantigen-binding fragment thereof interacts with at least ten amino acidscontained within SEQ ID NO:
 405. 18. The method of claim 15, wherein theantibody or antigen-binding fragment thereof interacts with at leasttwenty amino acids contained within SEQ ID NO:
 405. 19. The method ofclaim 14, wherein the antibody or antigen-binding fragment thereofinteracts with the amino acid sequence of SEQ ID NO:
 405. 20. The methodof claim 1, wherein the maytansinoid is DM4
 21. The method of claim 1,wherein the maytansinoid is DM1.
 22. The method of claim 1, wherein themaytansinoid is conjugated to the antibody via a cleavable linker. 23.The method of claim 1, wherein the maytansinoid is conjugated to theantibody via a non-cleavable linker.
 24. The method of claim 1, whereinthe maytansinoid is conjugated to the antibody via a linker selectedfrom the group consisting of 4-[-maleimidomethyl]cyclohexane-1-carboxylate), SPDB, mc-val-cit, and mc-val-cit-PAB. 25.The method of claim 1, wherein the maytansinoid is conjugated to theantibody via a linker, and wherein the linker is 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (MCC).
 26. The method of claim 1, wherein theADC inhibits prolactin mediated STAT5 activity.
 27. The method of claim1, wherein the ADC induces mitotic arrest in cells expressing PRLR. 28.The method of claim 1, wherein the ADC inhibits the growth of cellsexpressing PRLR.
 29. The method of claim 1, wherein the patient waspreviously treated with an estrogen receptor inhibitor.
 30. The methodof claim 1, wherein the effective amount is sufficient to delay orinhibit progression of the cancer.
 31. The method of claim 1, whereinthe ADC is administered intravenously.
 32. The method of claim 1,wherein the expression of PRLR in the breast cancer is not substantiallyreduced after treatment.
 33. The method of claim 1, wherein the ADCkills cells that express PRLR at an expression level of less than30-fold above background.
 34. The method of claim 1, wherein the ADCkills cells that express PRLR at an expression level of less than20-fold above background.
 35. The method of claim 1, wherein the ADCkills cells that express PRLR at an expression level greater than12-fold above background but less than 30-fold above background.
 36. Themethod of claim 1, wherein the antibody or antigen-binding fragmentthereof comprises a heavy chain variable region (HCVR)/light chainvariable region (LCVR) amino acid sequence pair selected from the groupconsisting of SEQ ID NOs: 18/26; 66/74; 274/282; 290/298; and 370/378.37. The method of claim 1, wherein the antibody or antigen-bindingfragment thereof comprises a heavy chain complementarity determiningregion (HCDR)-1 comprising SEQ ID NO: 20; an HCDR2 comprising SEQ ID NO:22; an HCDR3 comprising SEQ ID NO: 24; a light chain complementaritydetermining region (LCDR)-1 comprising SEQ ID NO: 28; an LCDR2comprising SEQ ID NO: 30; and an LCDR3 comprising SEQ ID NO:
 32. 38. Themethod of claim 37, wherein the antibody or antigen-binding fragmentthereof comprises an HCVR comprising SEQ ID NO: 18 and a LCVR comprisingSEQ ID NO:
 26. 39. The method of claim 1, wherein the antibody orantigen-binding fragment thereof comprises an HCDR1 comprising SEQ IDNO: 68; an HCDR2 comprising SEQ ID NO: 70; an HCDR3 comprising SEQ IDNO: 72; an LCDR1 comprising SEQ ID NO: 76; an LCDR2 comprising SEQ IDNO: 78; and an LCDR3 comprising SEQ ID NO:
 80. 40. The method of claim39, wherein the antibody or antigen-binding fragment thereof comprisesan HCVR comprising SEQ ID NO: 66 and a LCVR comprising SEQ ID NO: 74.41. The method of claim 1, wherein the antibody or antigen-bindingfragment thereof comprises an HCDR1 comprising SEQ ID NO: 276; an HCDR2comprising SEQ ID NO: 278; an HCDR3 comprising SEQ ID NO: 280; an LCDR1comprising SEQ ID NO: 284; an LCDR2 comprising SEQ ID NO: 286; and anLCDR3 comprising SEQ ID NO:
 288. 42. The method of claim 41, wherein theantibody or antigen-binding fragment thereof comprises an HCVRcomprising SEQ ID NO: 274 and a LCVR comprising SEQ ID NO:
 282. 43. Themethod of claim 1, wherein the antibody or antigen-binding fragmentthereof comprises an HCDR1 comprising SEQ ID NO: 292; an HCDR2comprising SEQ ID NO: 294; an HCDR3 comprising SEQ ID NO: 296; an LCDR1comprising SEQ ID NO: 300; an LCDR2 comprising SEQ ID NO: 302; and anLCDR3 comprising SEQ ID NO:
 304. 44. The method of claim 43, wherein theantibody or antigen-binding fragment thereof comprises an HCVRcomprising SEQ ID NO: 290 and a LCVR comprising SEQ ID NO:
 298. 45. Themethod of claim 1, wherein the antibody or antigen-binding fragmentthereof comprises an HCDR1 comprising SEQ ID NO: 372; an HCDR2comprising SEQ ID NO: 374; an HCDR3 comprising SEQ ID NO: 376; an LCDR1comprising SEQ ID NO: 380; an LCDR2 comprising SEQ ID NO: 382; and anLCDR3 comprising SEQ ID NO:
 384. 46. The method of claim 45, wherein theantibody or antigen-binding fragment thereof comprises an HCVRcomprising SEQ ID NO: 370 and a LCVR comprising SEQ ID NO:
 378. 47. Themethod of claim 1, wherein the antibody or antigen-binding fragmentthereof competes for binding to PRLR with a reference antibodycomprising an HCVR/LCVR amino acid sequence pair selected from the groupconsisting of SEQ ID NOs: 18/26; 66/74; 274/282; 290/298; and 370/378.48. A method of treating PRLR positive breast cancer in a patient, themethod comprising co-administering to a patient a therapeuticallyeffective amount of: (a) an antibody-drug conjugate (ADC) comprising anantibody or antigen-binding fragment thereof conjugated to DM1 throughan MCC linker, wherein the antibody or antigen-binding fragment thereofbinds to human prolactin receptor (PRLR), and (b) fulvestrant.
 49. Themethod of claim 48, wherein the antibody or antigen-binding fragmentthereof comprises an HCDR1 comprising SEQ ID NO: 292; an HCDR2comprising SEQ ID NO: 294; an HCDR3 comprising SEQ ID NO: 296; an LCDR1comprising SEQ ID NO: 300; an LCDR2 comprising SEQ ID NO: 302; and anLCDR3 comprising SEQ ID NO:
 304. 50. The method of claim 49, wherein theantibody or antigen-binding fragment thereof comprises an HCVR/LCVRamino acid sequence pair of SEQ ID NOs: 290/298.
 51. A method forkilling tumor cells that express low levels of PRLR, the methodcomprising contacting the cells with: (a) an antibody-drug conjugate(ADC) comprising an antibody or antigen-binding fragment thereofconjugated to a maytansinoid, wherein the antibody or antigen-bindingfragment thereof binds to human prolactin receptor (PRLR), and (b)fulvestrant.
 52. The method of claim 51, wherein the tumor cells expressless than 1 million copies of PRLR per cell.
 53. The method of claim 52,wherein the tumor cells express between 3000 and 500,000 copies of PRLRper cell.
 54. The method of claim 51, wherein the antibody orantigen-binding fragment thereof comprises an HCDR1 comprising SEQ IDNO: 292; an HCDR2 comprising SEQ ID NO: 294; an HCDR3 comprising SEQ IDNO: 296; an LCDR1 comprising SEQ ID NO: 300; an LCDR2 comprising SEQ IDNO: 302; and an LCDR3 comprising SEQ ID NO:
 304. 55. The method of claim54, wherein the antibody or antigen-binding fragment thereof comprisesan HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 290/298.
 56. Themethod of claim 51, wherein the maytansinoid is DM1.
 57. The method ofclaim 56, wherein the antibody or antigen-binding fragment thereof isconjugated to DM1 via a linker, and wherein the linker is4-(N-maleimidomethyl) cyclohexane-1-carboxylate (MCC).